Methods for detection of the Ob receptor and the diagnosis of body weight disorders, including obesity and cachexia

ABSTRACT

The present invention relates to the discovery, identification and characterization of nucleotides that encode Ob receptor (ObR), a receptor protein that participates in mammalian body weight regulation. The invention encompasses obR nucleotides, host cell expression systems, ObR proteins, fusion proteins, polypeptides and peptides, antibodies to the receptor, transgenic animals that express an obR transgene, or recombinant knock-out animals that do not express the ObR, antagonists and agonists of the receptor, and other compounds that modulate obR gene expression or ObR activity that can be used for diagnosis, drug screening, clinical trial monitoring, and/or the treatment of body weight disorders, including but not limited to obesity, cachexia and anorexia.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 09/950,149,filed Sep. 10, 2001, now U.S. Pat. No. 6,977,240, which is acontinuation of application Ser. No. 09/069,781, filed Apr. 29, 1998,issued as U.S. Pat. No. 6,287,782, which is a continuation-in-part ofapplication Ser. No. 08/864,564, filed May 28, 1997, now U.S. Pat. No.6,395,498 which is a continuation-in-part of application Ser. No.08/708,123, filed Sep. 3, 1996 issued as U.S. Pat. No. 6,482,927, whichis a continuation-in-part of application Ser. No. 08/638,524, filed Apr.26, 1996, issued as U.S. Pat. No. 6,548,269, which is acontinuation-in-part of U.S. application Ser. No. 08/599,455, filed Jan.22, 1996, issued as U.S. Pat. No. 5,972,621, which is acontinuation-in-part of application Ser. No. 08/583,153, filed Dec. 28,1995, issued as U.S. Pat. No. 6,506,877, which is a continuation-in-partof application Ser. No. 08/570,142, filed Dec. 11, 1995, issued as U.S.Pat. No. 6,509,189, which is a continuation-in-part of application Ser.No. 08/569,485, filed Dec. 8, 1995, now abandoned, which is acontinuation-in-part of application Ser. No. 08/566,622, filed Dec. 4,1995, now abandoned, which is a continuation-in-part of application Ser.No. 08/562,663, filed Nov. 27, 1995, now abandoned; all of which arehereby incorporated by reference in their entirety.

1. INTRODUCTION

The present invention relates to the discovery, identification andcharacterization of nucleotides that encode Ob receptor (ObR), areceptor protein that participates in mammalian body weight regulation.The invention encompasses obR nucleotides, host cell expression systems,ObR proteins, fusion proteins, polypeptides and peptides, antibodies tothe receptor, transgenic animals that express an obR transgene, orrecombinant knock-out animals that do not express the ObR, antagonistsand agonists of the receptor, and other compounds that modulate obR geneexpression or ObR activity that can be used for diagnosis, drugscreening, clinical trial monitoring, and/or the treatment of bodyweight disorders, including but not limited to obesity, cachexia andanorexia.

2. BACKGROUND OF THE INVENTION

Obesity represents the most prevalent of body weight disorders, and itis the most important nutritional disorder in the western world, withestimates of its prevalence ranging from 30% to 50% within themiddle-aged population. Other body weight disorders, such as anorexianervosa and bulimia nervosa which together affect approximately 0.2% ofthe female population of the western world, also pose serious healththreats. Further, such disorders as anorexia and cachexia (wasting) arealso prominent features of other diseases such as cancer, cysticfibrosis, and AIDS.

Obesity, defined as an excess of body fat relative to lean body mass,also contributes to other diseases. For example, this disorder isresponsible for increased incidences of diseases such as coronary arterydisease, stroke, and diabetes. (See, e.g., Nishina, P. M. et al., 1994,Metab. 43:554-558.) Obesity is not merely a behavioral problem, i.e.,the result of voluntary hyperphagia. Rather, the differential bodycomposition observed between obese and normal subjects results fromdifferences in both metabolism and neurologic/metabolic interactions.These differences seem to be, to some extent, due to differences in geneexpression, and/or level of gene products or activity (Friedman, J. M.et al., 1991, Mammalian Gene 1:130-144).

The epidemiology of obesity strongly shows that the disorder exhibitsinherited characteristics (Stunkard, 1990, N. Eng. J. Med. 322:1483).Moll et al. have reported that, in many populations, obesity seems to becontrolled by a few genetic loci (Moll et al. 1991, Am. J. Hum. Gen.49:1243). In addition, human twin studies strongly suggest a substantialgenetic basis in the control of body weight, with estimates ofheritability of 80-90% (Simopoulos, A. P. & Childs B., eds., 1989, InGenetic Variation and Nutrition in Obesity, World Review of Nutritionand Diabetes 63, S. Karger, Basel, Switzerland; Borjeson, M., 1976,Acta. Paediatr. Scand. 65:279-287).

Studies of non-obese persons who deliberately attempted to gain weightby systematically over-eating were found to be more resistant to suchweight gain and able to maintain an elevated weight only by very highcaloric intake. In contrast, spontaneously obese individuals are able tomaintain their status with normal or only moderately elevated caloricintake. In addition, it is a commonplace experience in animal husbandrythat different strains of swine, cattle, etc., have differentpredispositions to obesity. Studies of the genetics of human obesity andof models of animal obesity demonstrate that obesity results fromcomplex defective regulation of both food intake, food induced energyexpenditure and of the balance between lipid and lean body anabolism.

There are a number of genetic diseases in man and other species whichfeature obesity among their more prominent symptoms, along with,frequently, dysmorphic features and mental retardation. For example,Prader-Willi syndrome (PWS) affects approximately 1 in 20,000 livebirths, and involves poor neonatal muscle tone, facial and genitaldeformities, and generally obesity.

In addition to PWS, many other pleiotropic syndromes which includeobesity as a symptom have been characterized. These syndromes are moregenetically straightforward, and appear to involve autosomal recessivealleles. The diseases, which include, among others, Ahlstroem,Carpenter, Bardet-Biedl, Cohen, and Morgagni-Stewart-Monel Syndromes.

A number of models exist for the study of obesity (see, e.g., Bray, G.A., 1992, Prog. Brain Res. 93:333-341, and Bray, G. A., 1989, Amer. J.Clin. Nutr. 5:891-902). For example, animals having mutations which leadto syndromes that include obesity symptoms have been identified, andattempts have been made to utilize such animals as models for the studyof obesity. The best studied animal models, to date, for genetic obesityare mice models. For reviews, see for example, Friedman, J. M. et al.,1991, Mamm. Gen. 1:130-144; Friedman, J. M. and Liebel, R. L., 1992,Cell 69:217-220.

Studies utilizing mice have confirmed that obesity is a very complextrait with a high degree of heritability. Mutations at a number of locihave been identified which lead to obese phenotypes. These include theautosomal recessive mutations obese (ob), diabetes (db), fat (fat) andtubby (tub). In addition, the autosomal dominant mutations Yellow at theagouti locus and Adipose (Ad) have been shown to contribute to an obesephenotype.

The ob and db mutations are on chromosomes 6 and 4, respectively, butlead to a complex, clinically similar phenotype of obesity, evidentstarting at about one month of age, which includes hyperphagia, severeabnormalities in glucose and insulin metabolism, very poorthermoregulation and non-shivering thermogenesis, and extreme torpor andunderdevelopment of the lean body mass. This complex phenotype has madeit difficult to identify the primary defect attributable to themutations (Bray G. A., et al., 1989 Amer. J. Clin. Nutr. 5:891-902).

Using molecular and classical genetic markers, the db gene has beenmapped to midchromosome 4 (Friedman et al., 1991, Mamm. Gen. 1:130-144).The mutation maps to a region of the mouse genome that is syntonic withhuman, suggesting that, if there is a human homolog of db, it is likelyto map to human chromosome 1p.

The ob gene and its human homologue have recently been cloned (Zhang, Y.et al., 1994, Nature 372:425-432). The gene appears to produce a 4.5 kbadipose tissue messenger RNA which contains a 167 amino acid openreading frame. The predicted amino acid sequence of the ob gene productindicates that it is a secreted protein and may, therefore, play a roleas part of a signalling pathway from adipose tissue which may serve toregulate some aspect of body fat deposition. Further, recent studieshave shown that recombinant Ob protein, also known as leptin, whenexogenously administered, can at least partially correct theobesity-related phenotype exhibited by ob mice (Pelleymounter, M. A. etal., 1995, Science 269:540-543; Halalas, J. L. et al., 1995, Science269:543-546; Campfield, L. A. et al., 1995, Science 269:546-549). Recentstudies have suggested that obese humans and rodents (other than ob/obmice) are not defective in their ability to produce ob mRNA or protein,and generally produce higher levels than lean individuals (Maffei etal., 1995, Nature Med. 1 (11):1155-1161; Considine et al., 1995, J.Clin. Invest. 95 (6):2986-2988; Lohnqvist et al., 1995, Nature Med.1:950-953; Hamilton et al., 1995, Nature Med. 1:953-956). These datasuggest that resistance to normal or elevated levels of Ob may be moreimportant than inadequate Ob production in human obesity. However, thereceptor for the ob gene product, thought to be expressed in thehypothalamus, remains elusive.

Homozygous mutations at either the fat or tub loci cause obesity whichdevelops more slowly than that observed in ob and db mice (Coleman, D.L., and Eicher, E. M., 1990, J. Heredity 81:424-427), with tub obesitydeveloping slower than that observed in fat animals. This feature of thetub obese phenotype makes the development of tub obese phenotype closestin resemblance to the manner in which obesity develops in humans. Evenso, however, the obese phenotype within such animals can becharacterized as massive in that animals eventually attain body weightswhich are nearly two times the average weight seen in normal mice.

The fat mutation has been mapped to mouse chromosome 8, while the tubmutation has been mapped to mouse chromosome 7. According to Naggert etal., the fat mutation has recently been identified (Naggert, J. K., etal., 1995, Nature Genetics 10:135-141). Specifically, the fat mutationappears to be a mutation within the Cpe locus, which encodes thecarboxypeptidase (Cpe) E protein. Cpe is an exopeptidase involved in theprocessing of prohormones, including proinsulin.

The dominant Yellow mutation at the agouti locus, causes a pleiotropicsyndrome which causes moderate adult onset obesity, a yellow coat color,and a high incidence of tumor formation (Herberg, L. and Coleman, D. L.,1977, Metabolism 26:59), and an abnormal anatomic distribution of bodyfat (Coleman, D. L., 1978, Diabetologia 14:141-148). This mutation mayrepresent the only known example of a pleiotropic mutation that causesan increase, rather than a decrease, in body size. The mutation causesthe widespread expression of a protein which is normally seen only inneonatal skin (Michaud, E. J. et al., 1994, Genes Devel. 8:1463-1472).

Other animal models include fa/fa (fatty) rats, which bear manysimilarities to the ob/ob and db/db mice, discussed above. Onedifference is that, while fa/fa rats are very sensitive to cold, theircapacity for non-shivering thermogenesis is normal. Torpor seems to playa larger part in the maintenance of obesity in fa/fa rats than in themice mutants. In addition, inbred mouse strains such as NZO mice andJapanese KK mice are moderately obese. Certain hybrid mice, such as theWellesley mouse, become spontaneously fat. Further, several desertrodents, such as the spiny mouse, do not become obese in their naturalhabitats, but do become so when fed on standard laboratory feed.

Animals which have been used as models for obesity have also beendeveloped via physical or pharmacological methods. For example,bilateral lesions in the ventromedial hypothalamus (VMH) andventrolateral hypothalamus (VLH) in the rat are associated,respectively, with hyperphagia and gross obesity and with aphagia,cachexia and anorexia. Further, it has been demonstrated that feedingmonosodium-glutamate (MSG) or gold thioglucose to newborn mice alsoresults in an obesity syndrome.

Each of the rodent obesity models is accompanied by alterations incarbohydrate metabolism resembling those in Type II diabetes in man. Forexample, from both ob and db, congenic C57BL/KS mice develop a severediabetes with ultimate β cell necrosis and islet atrophy, resulting in arelative insulinopenia, while congenic C57BL/6J ob and db mice develop atransient insulin-resistant diabetes that is eventually compensated by βcell hypertrophy resembling human Type II diabetes.

With respect to ob and db mice, the phenotype of these mice resembleshuman obesity in ways other than the development of diabetes, in thatthe mutant mice eat more and expend less energy than do lean controls(as do obese humans). This phenotype is also quite similar to that seenin animals with lesions of the ventromedial hypothalamus, which suggeststhat both mutations may interfere with the ability to properly integrateor respond to nutritional information within the central nervous system.Support for this hypothesis comes from the results of parabiosisexperiments (Coleman, D. L. 1973, Diabetologica 9:294-298) that suggestob mice are deficient in a circulating satiety factor and that db miceare resistant to the effects of the ob factor. These experiments haveled to the conclusion that obesity in these mutant mice may result fromdifferent defects in an afferent loop and/or integrative center of thepostulated feedback mechanism that controls body composition.

In summary, therefore, obesity, which poses a major, worldwide healthproblem, represents a complex, highly heritable trait. Given theseverity, prevalence and potential heterogeneity of such disorders,there exists a great need for the identification of those genes and geneproducts that participate in the control of body weight.

It is an objective of the invention to provide modulators of bodyweight, to provide methods for diagnosis of body weight disorders, toprovide therapy for such disorders, and to provide assay systems for thescreening of substances that can be used to control body weight.

3. SUMMARY OF THE INVENTION

The present invention relates to the discovery, identification andcharacterization of nucleotides that encode Ob receptor (ObR), a novelreceptor protein that participates in the control of mammalian bodyweight. ObR, described for the first time herein, is a transmembraneprotein that spans the cellular membrane once and is involved in signaltransduction triggered by the binding of its natural ligand, Ob, alsoknown as leptin. ObR has amino acid sequence motifs found in the Class Icytokine receptor family, and is most related to the gp130 signaltransducing component of the IL-6 receptor, the G-CSF receptor, and theLIF receptor. The results presented in the working examples hereindemonstrate that a long-form ObR (predominantly expressed in thehypothalamus) transduces signal via a STAT mediated pathway typical ofIL-6 type cytokine receptors, whereas a major naturally occurringtruncated form or a mutant form found in obese db/db mice does not. Thelong form ObR can mediate activation of STAT proteins and stimulatetranscription through IL-6 responsive gene elements. Reconstitutionexperiments indicate that, although ObR mediates intracellular signalswith a specificity similar to IL-6 type cytokine receptors, signalingappears to be independent of the gp130 signal transducing component ofthe IL-6 type cytokine receptors.

The ObR mRNA transcript, which is about 5 kb long, is expressed in thechoroid plexus, the hypothalamus and other tissues, including lung andliver. The murine short forms described herein encode receptor proteinsof 894 (FIG. 1) and 893 amino acids; murine long form obR cDNAs andhuman obR cDNAs, described herein, encode receptor proteins of 1162amino acids and 1165 amino acids, respectively (FIG. 6 and FIG. 3,respectively). The ObR has a typical hydrophobic leader sequence (about22 amino acids long in both forms of murine ObR, and about 20 aminoacids long in human ObR); an extracellular domain (about 815 amino acidslong in both forms of murine ObR, and about 819 amino acids long inhuman ObR); a short transmembrane region (about 23 amino acids long inboth forms of murine ObR and human ObR); and a cytoplasmic domain. Thetranscripts encoding the murine ObR short (FIG. 1) and long form (FIG.6) are identical until the fifth codon 5′ of the stop codon of the shortform and then diverge completely, suggestive of alternative splicing. Asdescribed herein, the cytoplasmic domain encoded by the 894 amino acidmurine short form obR cDNA is 34 amino acids, while that encoded by themurine long form obR cDNA (302 amino acids) is approximately the samelength as the cytoplasmic domain encoded by the human obR cDNA (303amino acids). The deduced amino acid sequences from murine long form ObRand human ObR are homologous throughout the length of the coding regionand share 75% identity (FIG. 7).

The obese phenotype of the db mouse results from a G→T transversion inthe obR gene. This transversion creates a splice donor site which inturn leads to aberrant processing of obR long form mRNA in db mutants.In db mutants this aberrant processing generates long form mRNAs whichencode a truncated ObR protein that is identical to the 894 amino acidshort form ObR. Like the short form ObR, the mutant long form ObR lacksmost of the cytoplasmic domain and is incapable of transducing a signalvia a STAT mediated pathway. The signalling competant long form ObR,which is absent in the db/db mice, is required for body weightmaintenance.

The invention encompasses the following nucleotides, host cellsexpressing such nucleotides, and the expression products of suchnucleotides: (a) nucleotides that encode mammalian ObRs, including thehuman ObR, and the obR gene product; (b) nucleotides that encodeportions of the ObR that correspond to its functional domains, and thepolypeptide products specified by such nucleotide sequences, includingbut not limited to the extracellular domain (ECD), the transmembranedomain (TM), and the cytoplasmic domain (CD); (c) nucleotides thatencode mutants of the ObR in which all or a part of one of the domainsis deleted or altered, and the polypeptide products specified by suchnucleotide sequences, including but not limited to soluble receptors inwhich all or a portion of the TM is deleted, and nonfunctional receptorsin which all or a portion of the CD is deleted; (d) nucleotides thatencode fusion proteins containing the ObR or one of its domains (e.g.,the extracellular domain) fused to another polypeptide.

The invention also encompasses agonists and antagonists of ObR,including small molecules, large molecules, mutant Ob proteins thatcompete with native Ob, and antibodies, as well as nucleotide sequencesthat can be used to inhibit obR gene expression (e.g., antisense andribozyme molecules, and gene or regulatory sequence replacementconstructs) or to enhance obR gene expression (e.g., expressionconstructs that place the obR gene under the control of a strongpromoter system), and transgenic animals that express an obR transgeneor “knock-outs” that do not express ObR.

In addition, the present invention encompasses methods and compositionsfor the diagnostic evaluation, typing and prognosis of body weightdisorders, including obesity and cachexia, and for the identification ofsubjects having a predisposition to such conditions. For example, obRnucleic acid molecules of the invention can be used as diagnostichybridization probes or as primers for diagnostic PCR analysis for theidentification of obR gene mutations, allelic variations and regulatorydefects in the obR gene. The present invention further provides fordiagnostic kits for the practice of such methods.

Further, the present invention also relates to methods for the use ofthe obR gene and/or obR gene products for the identification ofcompounds which modulate, i.e., act as agonists or antagonists, of obRgene expression and or obR gene product activity. Such compounds can beused as agents to control body weight and, in particular, as therapeuticagents for the treatment of body weight and body weight disorders,including obesity, cachexia and anorexia.

Still further, the invention encompasses methods and compositions forthe treatment of body weight disorders, including obesity, cachexia, andanorexia. Such methods and compositions are capable of modulating thelevel of obR gene expression and/or the level of obR gene productactivity.

This invention is based, in part, on the surprising discovery, after anextensive survey of numerous cell lines and tissues, of a high affinityreceptor for Ob in the choroid plexus of the brain, the identificationand cloning of obR cDNA from a library prepared from choroid plexusmRNA, characterization of its novel sequence, mapping the obR gene tothe same genetic interval in the mouse genome as the db gene maps, andcharacterization of the ObR as a transmembrane receptor of the Class Icytokine receptor family. obR mRNA was detected in other tissues,including the hypothalamus.

The full-length ObR, expressed predominantly in the hypothalamus signalstransduces through activation of STAT proteins and stimulation oftranscription through IL-6 responsive gene elements. The ability of thefull-length long form ObR to signal is in contrast to the naturallyoccurring truncated form or the mutant form found in db/db mice whichare unable to mediate signal transduction. The invention also includesforms of ObR lacking one or another of the intracellular domainsimportant for signalling and induction of gene expression.

In another aspect the invention features a method for identifyingcandidate therapeutic agents for the treatment of a body weightdisorder, comprising:

a) providing a cell which expresses a mammalian Ob receptor, the cellharboring a reporter construct, the reporter construct including asequence encoding a detectable protein (e.g., β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacZ (encoding β-galactosidase), alkaline phosphatase orxanthine guaninephosphoribosyltransferase (XGPRT)), the sequenceencoding the detectable protein being operably linked to a Ob receptorresponsive regulatory element (e.g., IL-6 RE or HRRE);

b) contacting the cell with a test compound;

c) measuring the expression of the detectable protein in the presence ofthe test compound;

d) wherein an increase or a decrease in the expression of the detectableprotein in the presence of the test compound compared to the absence ofthe test compound indicates that the test compound is a candidatetherapeutic agent for treatment of a body weight disorder.

The sequence encoding the detectable protein is operably linked to theOb receptor responsive regulatory element if expression of the proteinis altered by activation of the Ob receptor (e.g., activation caused bythe binding of leptin to the Ob receptor).

In other embodiments the contacting with the test compound takes placein the presence of an Ob receptor agonist or antagonist. The inventionalso features a compound identified using the method described above.

In another aspect the invention features a method of inducing weightloss in a mammal by administering to the mammal a compound (e.g., asmall molecule or an antibody) that activates the Ob receptor or the Obreceptor signalling pathway.

3.1. Definitions

As used herein, the following terms, whether used in the singular orplural, will have the meanings indicated:

-   -   Ob: means the Ob protein described in Zhang, Y. et al., 1994,        Nature 372:425-432, which is incorporated herein by reference in        its entirety, which is also known as leptin. Ob includes        molecules that are homologous to Ob or which bind to ObR. Ob        fusion proteins having an N-terminal alkaline phosphatase domain        are referred to herein as AP-Ob fusion proteins, while Ob fusion        proteins having a C-terminal alkaline phosphatase domain are        referred to herein as Ob-AP fusion proteins.    -   obR nucleotides or coding sequences: means nucleotide sequences        encoding ObR protein, polypeptide or peptide fragments of ObR        protein, or ObR fusion proteins. obR nucleotide sequences        encompass DNA, including genomic DNA (e.g. the obR gene) or        cDNA, or RNA.    -   ObR: means Ob receptor protein. Polypeptides or peptide        fragments of ObR protein are referred to as ObR polypeptides or        ObR peptides. Fusions of ObR, or ObR polypeptides or peptide        fragments to an unrelated protein are referred to herein as ObR        fusion proteins. A functional ObR refers to a protein which        binds Ob with high affinity in vivo or in vitro.    -   ECD: means “extracellular domain”.    -   TM: means “transmembrane domain”.    -   CD: means “cytoplasmic domain”.

4. DESCRIPTION OF THE FIGURES

FIGS. 1A-1D. Nucleotide sequence (SEQ ID NO:1) and deduced amino acidsequence (SEQ ID NO:2) of murine obR (short form) cDNA encoding murineshort form ObR protein (894 amino acids). The domains of short formmurine ObR are: signal sequence (amino acid residue 1 to about aminoacid residue 22), extracellular domain (from about amino acid residue 23to about amino acid residue 837), transmembrane domain (from about aminoacid residue 838 to about amino acid residue 860), and cytoplasmicdomain (from about amino acid residue 861 to about amino acid residue894). Potential N-linked glycosylation sites in the extracellular domainare indicated by asterisks above the first amino acid of the N-X-S andN-X-T motifs. Underscores indicate motifs conserved in the class Icytokine receptor family.

FIG. 2A. COS-7 cells transfected with the ObR cDNA were treated withvarious AP or AP-Ob fusion proteins at 1 nM (diluted in DMEM+10% FBS).Columns show the average of two binding determinations and error barsshow the difference between the two. 1) Unfused AP, 2) AP-Ob (mouse), 3)AP-Ob (mouse)+100 nM mouse Ob, 4) AP-Ob (mouse)+100 nM human Ob, 5)AP-Ob (human), 6) Ob-AP (mouse), 7) AP-Ob (mouse) incubated with mocktransfected (vector- no insert) COS-7 cells.

FIG. 2B. Binding isotherm and Scatchard analysis of the interaction ofAP-Ob and ObR. COS-7 cells transfected with the obR cDNA were incubatedwith various concentrations of the AP-Ob (mouse) fusion protein.Scatchard transformation is shown as an inset.

FIGS. 3A-3F. Nucleotide sequence (SEQ ID NO:3) and deduced amino acidsequence (SEQ ID NO:4) of human obR cDNA encoding human ObR protein. Thedomains of human ObR are: signal sequence (from amino acid residue 1 toabout amino acid residue 20). extracellular domain (from about aminoacid residue 21 to about amino arid residue 839), transmembrane domain(from about amino acid residue 840 to about amino acid residue 862), andcytoplasmic domain (from about amino acid residue 863 to about aminoacid residue 1165). Also depicted are 5′ untranslated nucleotidesequences. Potential N-linked glycosylation sites in the extracellulardomain are indicated by asterisks above the first amino acid of theN-X-S and N-X-T motifs. Underscores indicate motifs conserved in theclass I cytokine receptor family

FIG. 4. Alignment of the extracellular domains of the murine ObR andhuman gp130(SEQ ID NO:5). Identical residues (black) and conservativechanges (gray) are indicated by shading around the corresponding aminoacids. Conservative changes (as defined by FASTA) are indicated.

FIGS. 5A-5B. Alignment of mouse ObR (short form shown in FIG. 1. SEQ IDNO:2) and human ObR (SEQ ID NO:4). Amino acids that are identicalbetween the two sequences are indicated by a star.

FIGS. 6A-6F. Nucleotide sequence and deduced amino acid sequence ofmurine long form obR cDNA (SEQ ID NO:42) encoding murine long form ObRprotein (SEQ ID NO:43). The domains of long form murine ObR are: signalsequence (amino acid residue 1 to about amino acid residue 22),extracellular domain (from about amino acid residue 23 to about aminoacid residue 837), transmembrane domain (from about amino acid residue838 to about amino acid residue 860), and cytoplasmic domain (from aboutamino acid residue 861 to about amino acid residue 1162).

FIGS. 7A-7B. Alignment of the long forms of human (SEQ ID NO:4) andmurine (SEQ ID NO:43) ObR. Identical residues and conservative changesare indicated by two asterisks or one asterisk, respectively.Conservative changes indicated are as defined by FASTA. Abbreviations:mobr-1, murine ObR long form; and hobr, human homolog.

FIG. 8. Location of the gene encoding ObR on mouse chromosome 4.

FIG. 9. Nucleotide sequence of the 106 base pair insert in the long formtranscript of db/db. The precise position of the insertion in thededuced amino acid sequence near the insertion region are shown (see SEQID NOs: 1, 2, 42 and 43, per section 8.2.2).

FIG. 10. Bar graph depicting ObR-Ig neutralization of OB protein. COScell were transiently transfected with the ObR cDNA and tested for theirability to bind 0.5 nM AP-OB. Column 1 shows the high levels of specificbinding observed in the absence of ObR-IgG fusion protein. Columns 2, 3,and 4 show the near complete inhibition of binding observed with threedifferent column fractions of purified ObR IgG.

FIG. 11A. Schematic drawings of various C-terminal deletion mutants ofObR protein. The names and predicted length (aa) of the proteins areshown above each protein. The extracellular domains are shown asstriped, the transmembrane domains are shown as black, and thecytoplasmic domains are shown as white. The location of tyrosineresidues in the cytoplasmic domain are indicated by horizontal bars(Y986, Y1079, and Y1141 are conserved between human and murine ObR). Thelength of the cytoplasmic domains (aa) are shown below each protein.

FIG. 11B. A bar graph depicting the results of CAT assays employing anIL-6RE-CAT expression construct (upper panel) or a HRRE-CAT expressionconstruct (lower panel) and the ObR deletion mutants of FIG. 11A. H-35cells were transfected with cDNAs encoding the ObR mutant and eitherIL-6RE-CAT or HRRE-CAT. Subcultures of cells were treated for 24 hourswith serum-free medium alone (−) or serum-free medium containing mouseleptin (+). CAT activity was determined and is expressed relative tovalues obtained for untreated control cultures.

FIG. 12. A bar graph depicting the results of an AP-Ob fusion proteinbinding assay. COS-7 cells were transfected with a cDNA encoding theindicated ObR protein. Forty-eight hours later, cells were incubatedwith 1 mM AP-Ob fusion protein. Bars show the average of two bindingassays. The error bars indicate the difference between the two assays.

FIG. 13A. Schematic drawings of various mutant ObR proteins. Thelocation of tyrosine residues 986 and 1079 are indicated. The locationof the “box 1” sequence is also indicated.

FIG. 13B. Bar graph depicting the results of a HRRE-CAT induction assay.H-35 cells were co-transfected with HRRE-CAT and expression constructsfor either OB-RY986F, OB-RY1079F or OB-R(box 1mt). Subcultures of cellswere treated for 24 hours with serum-free medium containing humanleptin. CAT activity was determined and is expressed relative to valuesobtained for untreated control cultures.

FIG. 14A. Schematic drawings of various receptor chimeras. The portionsderived from G-CSFR are shaded; the portions derived from ObR are not.The locations of the predicted Box 1, Box 2, and Box 3 motifs areindicated.

FIG. 14B. Bar graphs depicting the results of IL-6RE-CAT (left panel)and HRRE-CAT induction assays. H-35 cells were co-transfected withexpression plasmids for the indicated receptor (ObR, G-CSFR, orchimeric) and IL-6-RE-CAT or HREE-CAT expression construct. Cells werestimulated with the appropriate ligand and CAT activity was determinedas in the experiments described in FIG. 11. All values are expressedrelative to untreated control cultures (mean±std deviation of 3 to 4experiments).

FIG. 15A. Bar graph depicting the results of HRRE-CAT induction assays.H-35 cells were co-transfected with HRRE-CAT and the indicated amount ofObR and OB-RΔ868-1165. Cells were stimulated with leptin, and CATactivity was determined as in the experiments described in FIG. 11. Allvalues are expressed relative to the untreated cultures. FIG. 15B. Bargraph depicting the results of IL-6RE-CAT induction assays. H-35 cellswere co-transfected with IL-6RE-CAT and the indicated amount ofObR/G-CSFR and OB-RA868-1165. Cells were stimulated with leptin, and CATactivity was determined as in the experiments described in FIG. 11. Allvalues are expressed relative to the untreated cultures.

FIG. 15C. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of G-CSFR and G-CSFR(Δcyto). Cells were stimulated with G-CSF,and CAT activity was determined as in the experiments described in FIG.11. All values are expressed relative to the untreated cultures.

FIG. 15D. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of G-CSFR/ObR and G-CSFR(Δcyto). Cells were stimulated withG-CSF, and CAT activity was determined as in the experiments describedin FIG. 11. All values are expressed relative to the untreated cultures.

FIG. 15E. Bar graph depicting the results of IL-6RE-CAT inductionassays. H-35 cells were co-transfected with IL-6RE-CAT and the indicatedamount of ObR and OB-RY1141F. Cells were stimulated with leptin, and CATactivity was determined as in the experiments described in FIG. 11. Allvalues are expressed relative to the untreated cultures.

FIG. 16. Line graph depicting % food intake over time in mice treatedwith PBS (□), an Ob receptor-Ig fusion protein (150 μg/mouse; •), LPS(10 μg/mouse; ◯), and LPS (10 μg/mouse) with the Ob receptor-Ig fusionprotein (150 μg/mouse) (_).

5. DETAILED DESCRIPTION OF THE INVENTION

ObR, described for the first time herein, is a novel receptor proteinthat participates in body weight regulation. ObR is a transmembraneprotein that spans the membrane once and belongs to the Class I familyof cytokine receptors, and is most closely related to the gp130 signaltransducing component of the IL-6 receptor, the G-CSF receptor, and theLIF receptor. Signal transduction is triggered by the binding of Ob tothe receptor. Neutralization of Ob, removal of Ob, or interference withits binding to ObR results in weight gain. ObR mRNA is detected in thechoroid plexus, and other tissues, including the hypothalamus.

The invention encompasses the use of obR nucleotides, ObR proteins andpeptides, as well as antibodies to the ObR (which can, for example, actas ObR agonists or antagonists), antagonists that inhibit receptoractivity or expression, or agonists that activate receptor activity orincrease its expression in the diagnosis and treatment of body weightdisorders, including, but not limited to obesity, cachexia and anorexiain animals, including humans. The diagnosis of an ObR abnormality in apatient, or an abnormality in the ObR signal transduction pathway, willassist in devising a proper treatment or therapeutic regimen. Inaddition, obR nucleotides and ObR proteins are useful for theidentification of compounds effective in the treatment of body weightdisorders regulated by the ObR.

In particular, the invention described in the subsections belowencompasses ObR, polypeptides or peptides corresponding to functionaldomains of the ObR (e.g., ECD, TM or CD), mutated, truncated or deletedObRs (e.g. an ObR with one or more functional domains or portionsthereof deleted, such as ΔTM and/or ΔCD), ObR fusion proteins (e.g. anObR or a functional domain of ObR, such as the ECD, fused to anunrelated protein or peptide such as an immunoglobulin constant region,i.e., IgFc), nucleotide sequences encoding such products, and host cellexpression systems that can produce such ObR products.

The invention also features Ob receptors having an amino acid sequencethat is substantially identical to a defined amino acid sequence.

By “substantially identical” is meant a polypeptide or nucleic acidhaving a sequence that is at least 85%, preferably 90%, and morepreferably 95% or more identical to the sequence of the reference aminoacid or nucleic acid sequence. For polypeptides, the length of thereference polypeptide sequence will generally be at least 16 aminoacids, preferably at least 20 amino acids, more preferably at least 25amino acids, and most preferably 35 amino acids. For nucleic acids, thelength of the reference nucleic acid sequence will generally be at least50 nucleotides, preferably at least 60 nucleotides, more preferably atleast 75 nucleotides, and most preferably 110 nucleotides.

Sequence identity can be measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705) with the default parameters specifiedtherein.

In the case of polypeptide sequences which are less than 100% identicalto a reference sequence, the non-identical positions are preferably, butnot necessarily, conservative substitutions for the reference sequence.Conservative substitutions typically include substitutions within thefollowing groups: glycine and alanine; valine, isoleucine, and leucine;aspartic acid and glutamic acid; asparagine and glutamine; serine andthreonine; lysine and arginine; and phenylalanine and tyrosine.

Where a particular polypeptide is said to have a specific percentidentity to a reference polypeptide of a defined length, the percentidentity is relative to the reference peptide. Thus, a peptide that is50% identical to a reference polypeptide that is 100 amino acids longcan be a 50 amino acid polypeptide that is completely identical to a 50amino acid long portion of the reference polypeptide. It might also be a100 amino acid long polypeptide which is 50% identical to the referencepolypeptide over its entire length. Of course, many other polypeptideswill meet the same criteria.

The invention also encompasses antibodies and anti-idiotypic antibodies(including Fab fragments), antagonists and agonists of the ObR, as wellas compounds or nucleotide constructs that inhibit expression of the obRgene (transcription factor inhibitors, antisense and ribozyme molecules,or gene or regulatory sequence replacement constructs), or promoteexpression of ObR (e.g., expression constructs in which obR codingsequences are operatively associated with expression control elementssuch as promoters, promoter/enhancers, etc.). The invention also relatesto host cells and animals genetically engineered to express the humanObR (or mutants thereof) or to inhibit or “knock-out” expression of theanimal's endogenous ObR.

The ObR proteins or peptides, ObR fusion proteins, obR nucleotidesequences, antibodies, antagonists and agonists can be useful for thedetection of mutant ObRs or inappropriately expressed ObRs for thediagnosis of body weight disorders such as obesity, anorexia orcachexia. The ObR proteins or peptides, ObR fusion proteins, obRnucleotide sequences, host cell expression systems, antibodies,antagonists, agonists and genetically engineered cells and animals canbe used for screening for drugs effective in the treatment of such bodyweight disorders. The use of engineered host cells and/or animals mayoffer an advantage in that such systems allow not only for theidentification of compounds that bind to the ECD of the ObR, but canalso identify compounds that affect the signal transduced by theactivated ObR.

Finally, the ObR protein products (especially soluble derivatives suchas peptides corresponding to the ObR ECD, or truncated polypeptideslacking the TM domain) and fusion protein products (especially ObR-Igfusion proteins, i.e., fusions of the ObR or a domain of the ObR, e.g.,ECD, ΔTM to an IgFc), antibodies and anti-idiotypic antibodies(including Fab fragments), antagonists or agonists (including compoundsthat modulate signal transduction which may act on downstream targets inthe ObR signal transduction pathway) can be used for therapy of suchdiseases. For example, the administration of an effective amount ofsoluble ObR ECD, ΔTM ObR or an ECD-IgFc fusion protein or ananti-idiotypic antibody (or its Fab) that mimics the ObR ECD would “mopup” or “neutralize” endogenous Ob, and prevent or reduce binding andreceptor activation, leading to weight gain. Nucleotide constructsencoding such ObR products can be used to genetically engineer hostcells to express such ObR products in vivo; these genetically engineeredcells function as “bioreactors” in the body delivering a continuoussupply of the ObR, ObR peptide, soluble ECD or ΔTM or ObR fusion proteinthat will “mop up” or neutralize Ob. Nucleotide constructs encodingfunctional ObRs, mutant ObRs, as well as antisense and ribozymemolecules can be used in “gene therapy” approaches for the modulation ofObR expression and/or activity in the treatment of body weightdisorders. Thus, the invention also encompasses pharmaceuticalformulations and methods for treating body weight disorders.

The invention is based, in part, on the surprising discovery of a highaffinity receptor for Ob expressed at significant concentration in thechoroid plexus. This discovery was made possible by using a novelalkaline phosphatase/Ob (AP-Ob) fusion protein for in situ staining ofcells and tissue. Competition studies with unlabeled Ob confirmed thatthe in situ binding observed was specific for Ob. Murine obR cDNA wasidentified using AP-Ob fusion protein to screen an expression library ofcDNAs synthesized from murine choroid plexus mRNA and transientlytransfected into mammalian COS cells. A clone, famj5312, expressing theshort form of a high affinity receptor for Ob was identified andsequenced. Sequence analysis revealed that the obR cDNA and predictedamino acid sequence are novel sequences containing amino acid regionsindicating that ObR is a member of the Class I family of receptorproteins. Mapping studies described herein demonstrate that the obR genemaps to the db locus. The data presented herein demonstrate further thatthe db gene is a mutant obR gene, which expresses an aberrantly splicedobR long form message that encodes a protein identical to the short formmurine ObR. The famj5312 sequence was utilized to screen a human fetalbrain cDNA library, which resulted in the identification of a human obRcDNA clone fahj5312d, described herein. Oligonucleotide primers designedon the basis of the human cDNA sequence were used to clone the humangenomic DNA clone, h-obR-p87, also described herein. mRNA encoding themurine long form of ObR was cloned from murine hypothalamus usingdegenerate primers designed on the human ObR cytoplasmic domain.

Various aspects of the invention are described in greater detail in thesubsections below.

5.1. The ObR Gene

The cDNA sequence (SEQ ID NO:1) and deduced amino acid sequence (SEQ IDNO:2) of murine short form (894 amino acids long) and murine long formObR are shown in FIGS. 1A-1D and 6A-6F, respectively. The signalsequence of both murine short and long form ObR extends from amino acidresidue 1 to about amino acid residue 22 of FIGS. 1 and 6, respectively;the extracellular domain of both forms of murine ObR extends from aboutamino acid residue 23 to about amino acid residue 837 of FIGS. 1A-1D and6A-6F; the transmembrane domain of both forms of murine ObR extends fromabout amino acid residue 838 to about amino acid residue 860 of FIGS.1A-1D and 6A-6F; and the cytoplasmic domain of the murine short form ObRextends from about amino acid residue 861 to about amino acid residue894 of FIG. 1, while that of the long form extends from amino acidresidue 861 to about amino acid residue 1162 of FIG. 6A-6F. At least oneother short form of murine ObR has been identified, which is one aminoacid shorter (i.e., 893 amino acids) than the sequence shown in FIG. 1.The sequence at the C-terminus differs from the sequence shown in FIG.1A-1D, in that residues 890-894 (RTDTL) are not present; and instead,residues 890-893 of the second short form have the following sequence:IMWI.

The cDNA sequence (SEQ ID NO:3) and deduced amino acid sequence (SEQ IDNO:4) of human ObR are shown in FIGS. 3A-3F. The human ObR signalsequence extends from amino acid residue 1 to about amino acid residue20 of FIGS. 3A-3F; the extracellular domain of human ObR extends fromabout amino acid residue 21 to about amino acid residue 839 of FIGS.3A-3F; the transmembrane domain of human ObR extends from about aminoacid residue 840 to about amino acid residue 862 of FIGS. 3A-3F; and thecytoplasmic domain of human ObR extends from about amino acid residue863 to about amino acid residue 1165 of FIGS. 3A-3F. Sequences derivedfrom the human cDNA clone were used to design primers that were used toclone the human genomic obR, h-obR -p87, as described in the examples,infra.

Data presented in the working examples, infra, demonstrate that the obRgene maps to the db locus, and that the db gene is a mutant obR genewhich is expressed in db mice as an aberrantly spliced transcriptresulting in an mRNA species containing an insert of approximately 106nucleotides (nt) in the portion encoding the cytoplasmic domain of ObR.The insert produces a mutation that results in a transcript that encodesa prematurely truncated long form that is identical to murine short formObR.

The obR nucleotide sequences of the invention include: (a) the DNAsequence shown in FIG. 1A-1D, 3A-3F or 6A-6F or contained in the cDNAclone famj5312 within E. coli strain 5312B4F3 as deposited with theAmerican Type Culture Collection (ATCC), or contained in the cDNA clonefahj5312d within E. coil strain h-obRD as deposited with the ATCC, orcontained in the human genomic clone, h-obR-p87 as deposited with theATCC; (b) nucleotide sequence that encodes the amino acid sequence shownin FIG. 1A-1D, 3A-3F or 6A-6F, or the ObR amino acid sequence encoded bythe cDNA clone famj5312 as deposited with the ATCC, or the cDNA clonefahj5312d as deposited with the ATCC, or contained in the human genomicclone, h-obR-p87 as deposited with the ATCC; (c) any nucleotide sequencethat hybridizes to the complement of the DNA sequence shown in FIG.1A-1D, 3A-3F or 6A-6F or contained in the cDNA clone famj5312 asdeposited with the ATCC, or contained in the cDNA clone fahj5312d asdeposited with the ATCC, or contained in the human genomic clone,h-obR-p87 as deposited with the ATCC under highly stringent conditions,for example, hybridization to filter-bound DNA in 0.5 M NaHPO₄, 7%sodium dodecyl sulfate (SDS), 1 mM EDTA at 65° C., and washing in0.1×SSC/0.1% SDS at 68° C. (Ausubel F. M. et al., eds., 1989, CurrentProtocols in Molecular Biology, Vol. I, Green Publishing Associates,Inc., and John Wiley & sons, Inc., New York, at p. 2.10.3) and encodes afunctionally equivalent gene product; and (d) any nucleotide sequencethat hybridizes to the complement of the DNA sequences that encode theamino acid sequence shown in FIG. 1A-1D, 3A-3F or 6A-6F contained incDNA clone famj5312 as deposited with the ATCC, or contained in the cDNAclone fahj5312d as deposited with the ATCC, or contained in the humangenomic clone, h-obR-p87 as deposited with the ATCC under less stringentconditions, such as moderately stringent conditions, for example,washing in 0.2×SSC/0.1% SDS at 42° C. (Ausubel et al., 1989, supra), yetwhich still encodes a functionally equivalent obR gene product.Functional equivalents of the ObR include naturally occurring ObRpresent in other species, and mutant ObRs whether naturally occurring orengineered. The invention also includes degenerate variants of sequences(a) through (d).

The invention also includes nucleic acid molecules, preferably DNAmolecules, that hybridize to, and are therefore the complements of, thenucleotide sequences (a) through (d), in the preceding paragraph. Suchhybridization conditions may be highly stringent or less highlystringent, as described above. In instances wherein the nucleic acidmolecules are deoxyoligonucleotides (“oligos”), highly stringentconditions may refer, for example, to washing in 6×SSC/0.05% sodiumpyrophosphate at 37° C. (for 14-base oligos), 48° C. (for 17-baseoligos), 55° C. (for 20-base oligos), and 60° C. (for 23-base oligos).These nucleic acid molecules may encode or act as obR antisensemolecules, useful, for example, in obR gene regulation (for and/or asantisense primers in amplification reactions of obR gene nucleic acidsequences). With respect to obR gene regulation, such techniques can beused to regulate, for example, cachexia and/or anorexia. Further, suchsequences may be used as part of ribozyme and/or triple helix sequences,also useful for obR gene regulation. Still further, such molecules maybe used as components of diagnostic methods whereby, for example, thepresence of a particular obR allele responsible for causing a weightdisorder, such as obesity, may be detected.

In addition to the obR nucleotide sequences described above, full lengthobR cDNA or gene sequences present in the same species and/or homologsof the obR gene present in other species can be identified and readilyisolated, without undue experimentation, by molecular biologicaltechniques well known in the art. The identification of homologs of obRin related species can be useful for developing animal model systemsmore closely related to humans for purposes of drug discovery. Forexample, expression libraries of cDNAs synthesized from choroid plexusmRNA derived from the organism of interest can be screened using labeledOb derived from that species, for example, an AP-Ob fusion protein.Alternatively, such cDNA libraries, or genomic DNA libraries derivedfrom the organism of interest can be screened by hybridization using thenucleotides described herein as hybridization or amplification probes.Furthermore, genes at other genetic loci within the genome that encodeproteins which have extensive homology to one or more domains of the obRgene product can also be identified via similar techniques. In the caseof cDNA libraries, such screening techniques can identify clones derivedfrom alternatively spliced transcripts in the same or different species.

Screening can be by filter hybridization, using duplicate filters. Thelabeled probe can contain at least 15-30 base pairs of the obRnucleotide sequence, as shown in FIG. 1A-1D, 3A-3F or 6A-6F. Thehybridization washing conditions used should be of a lower stringencywhen the cDNA library is derived from an organism different from thetype of organism from which the labeled sequence was derived. Withrespect to the cloning of a human obR homolog, using murine obR probes,for example, hybridization can, for example, be performed at 65° C.overnight in Church's buffer (7% SDS, 250 mM NaHPO₄, 2 μM EDTA, 1% BSA).Washes can be done with 2×SSC, 0.1% SDS at 65° C. and then at 0.1×SSC,0.1% SDS at 65° C.

Low stringency conditions are well known to those of skill in the art,and will vary predictably depending on the specific organisms from whichthe library and the labeled sequences are derived. For guidanceregarding such conditions see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, Cold Springs Harbor Press, N.Y.;and Ausubel et al., 1989, Current Protocols in Molecular Biology, GreenPublishing Associates and Wiley Interscience, N.Y.

Alternatively, the labeled obR nucleotide probe may be used to screen agenomic library derived from the organism of interest, again, usingappropriately stringent conditions. The identification andcharacterization of human genomic clones is helpful for designingdiagnostic tests and clinical protocols for treating body weightdisorders in human patients. For example, sequences derived from regionsadjacent to the intron/exon boundaries of the human gene can be used todesign primers for use in amplification assays to detect mutationswithin the exons, introns, splice sites (e.g. splice acceptor and/ordonor sites), etc., that can be used in diagnostics.

Further, an obR gene homolog may be isolated from nucleic acid of theorganism of interest by performing PCR using two degenerateoligonucleotide primer pools designed on the basis of amino acidsequences within the obR gene product disclosed herein. The template forthe reaction may be cDNA obtained by reverse transcription of mRNAprepared from, for example, human or non-human cell lines or tissue,such as choroid plexus, known or suspected to express an obR geneallele.

The PCR product may be subcloned and sequenced to ensure that theamplified sequences represent the sequences of an obR gene. The PCRfragment may then be used to isolate a full length cDNA clone by avariety of methods. For example, the amplified fragment may be labeledand used to screen a cDNA library, such as a bacteriophage cDNA library.Alternatively, the labeled fragment may be used to isolate genomicclones via the screening of a genomic library.

PCR technology may also be utilized to isolate full length cDNAsequences. For example, RNA may be isolated, following standardprocedures, from an appropriate cellular or tissue source (i.e., oneknown, or suspected, to express the obR gene, such as, for example,choroid plexus or brain tissue). A reverse transcription reaction may beperformed on the RNA using an oligonucleotide primer specific for themost 5′ end of the amplified fragment for the priming of first strandsynthesis. The resulting RNA/DNA hybrid may then be “tailed” withguanines using a standard terminal transferase reaction, the hybrid maybe digested with RNAase H, and second strand synthesis may then beprimed with a poly-C primer. Thus, cDNA sequences upstream of theamplified fragment may easily be isolated. For a review of cloningstrategies which may be used, see for example, Sambrook et al., 1989,supra.

The obR gene sequences may additionally be used to isolate mutant obRgene alleles. Such mutant alleles may be isolated from individualseither known or proposed to have a genotype which contributes to thesymptoms of body weight disorders such as obesity, cachexia or anorexia.Mutant alleles and mutant allele products may then be utilized in thetherapeutic and diagnostic systems described below. Additionally, suchobR gene sequences can be used to detect obR gene regulatory (e.g.,promoter or promotor/enhancer) defects which can affect body weight.

A cDNA of a mutant obR gene may be isolated, for example, by using PCR,a technique which is well known to those of skill in the art. In thiscase, the first cDNA strand may be synthesized by hybridizing anoligo-dT oligonucleotide to mRNA isolated from tissue known or suspectedto be expressed in an individual putatively carrying the mutant obRallele, and by extending the new strand with reverse transcriptase. Thesecond strand of the cDNA is then synthesized using an oligonucleotidethat hybridizes specifically to the 5′ end of the normal gene. Usingthese two primers, the product is then amplified via PCR, cloned into asuitable vector, and subjected to DNA sequence analysis through methodswell known to those of skill in the art. By comparing the DNA sequenceof the mutant obR allele to that of the normal obR allele, themutation(s) responsible for the loss or alteration of function of themutant obR gene product can be ascertained.

Alternatively, a genomic library can be constructed using DNA obtainedfrom an individual suspected of or known to carry the mutant obR allele,or a cDNA library can be constructed using RNA from a tissue known, orsuspected, to express the mutant obR allele. The normal obR gene or anysuitable fragment thereof may then be labeled and used as a probe toidentify the corresponding mutant obR allele in such libraries. Clonescontaining the mutant obR gene sequences may then be purified andsubjected to sequence analysis according to methods well known to thoseof skill in the art.

Additionally, an expression library can be constructed utilizing cDNAsynthesized from, for example, RNA isolated from a tissue known, orsuspected, to express a mutant obR allele in an individual suspected ofor known to carry such a mutant allele. In this manner, gene productsmade by the putatively mutant tissue may be expressed and screened usingstandard antibody screening techniques in conjunction with antibodiesraised against the normal obR gene product, as described, below, inSection 5.3. (For screening techniques, see, for example, Harlow, E. andLane, eds., 1988, Antibodies: A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor.) Additionally, screening can be accomplishedby screening with labeled Ob fusion proteins, such as, for example,AP-Ob or Ob-AP fusion proteins. In cases where an obR mutation resultsin an expressed gene product with altered function (e.g., as a result ofa missense or a frameshift mutation), a polyclonal set of antibodies toObR are likely to cross-react with the mutant ObR gene product. Libraryclones detected via their reaction with such labeled antibodies can bepurified and subjected to sequence analysis according to methods wellknown to those of skill in the art.

The invention also encompasses nucleotide sequences that encode mutantObRs, peptide fragments of the ObR, truncated ObRs, and ObR fusionproteins. These include, but are not limited to nucleotide sequencesencoding mutant ObRs described in section 5.2 infra; polypeptides orpeptides corresponding to the ECD, TM and/or CD domains of the ObR orportions of these domains; truncated ObRs in which one or two of thedomains is deleted, e.g., a soluble ObR lacking the TM or both the TMand CD regions, or a truncated, nonfunctional ObR lacking all or aportion of the CD region. Nucleotides encoding fusion proteins mayinclude by are not limited to full length ObR, truncated ObR or peptidefragments of ObR fused to an unrelated protein or peptide, such as forexample, a transmembrane sequence, which anchors the ObR ECD to the cellmembrane; an Ig Fc domain which increases the stability and half life ofthe resulting fusion protein (e.g., ObR-Ig) in the bloodstream; or anenzyme, fluorescent protein, luminescent protein which can be used as amarker.

The invention also encompasses (a) DNA vectors that contain any of theforegoing ObR coding sequences and/or their complements (i.e.,antisense); (b) DNA expression vectors that contain any of the foregoingObR coding sequences operatively associated with a regulatory elementthat directs the expression of the coding sequences; and (c) geneticallyengineered host cells that contain any of the foregoing ObR codingsequences operatively associated with a regulatory element that directsthe expression of the coding sequences in the host cell. As used herein,regulatory elements include but are not limited to inducible andnon-inducible promoters, enhancers, operators and other elements knownto those skilled in the art that drive and regulate expression. Suchregulatory elements include but are not limited to the cytomegalovirushCMV immediate early gene, the early or late promoters of SV40adenovirus, the lac system, the trp system, the TAC system, the TRCsystem, the major operator and promoter regions of phage A, the controlregions of fd coat protein, the promoter for 3-phosphoglycerate kinase,the promoters of acid phosphatase, and the promoters of the yeastα-mating factors.

5.2. ObR Proteins and Polypeptides

ObR protein, polypeptides and peptide fragments, mutated, truncated ordeleted forms of the ObR and/or ObR fusion proteins can be prepared fora variety of uses, including but not limited to the generation ofantibodies, as reagents in diagnostic assays, the identification ofother cellular gene products involved in the regulation of body weight,as reagents in assays for screening for compounds that can be used inthe treatment of body weight disorders, and as pharmaceutical reagentsuseful in the treatment of body weight disorders related to the ObR.

FIGS. 1A-1D and 6A-6F show the amino acid sequence of a murine shortform and long form ObR protein, respectively. In both of these forms ofObR, the signal sequence extends from amino acid 1 to about amino acid22; the ECD extends from about amino acid 23 to about amino acid 837;and the TM extends from about amino acid 838 to about amino acid 860. Inthe short form of murine ObR, the CD extends from about amino acid 861to about amino acid 894 (or to 893 in the second short form), while inthe long form it extends from about amino acid 861 to about amino acid1162. FIGS. 3A-3F show the amino acid sequence of a human ObR. Thesignal sequence extends from amino acid residue 1 to about amino acidresidue 20; the ECD extends from about amino acid residue 21 to aboutamino acid residue 839; the TM extends from about amino acid residue 840to about amino acid residue 862; and the CD extends from about aminoacid residue 863 to about amino acid residue 1165.

The ObR sequence begins with a methionine in a DNA sequence contextconsistent with a translation initiation site, followed by a typicalhydrophobic signal sequence of peptide secretion. The predicted matureextracellular domain for both forms of murine ObR is identical and is815 amino acids long, whereas the ECD predicted for human ObR is 819amino acids long. The extracellular domain of ObR shows many features ofthe class I cytokine receptor family (reviewed in Heldin, 1995, Cell80:213-223), and is most closely related to the gp130 signal transducingcomponent of the IL-6 receptor (Taga et at., 1989, Cell 58:573-581), theG-CSF receptor (Fukunaga et al., 1990, Cell 61:341-350), and the LIFreceptor (Gearing et al., 1991, Science 255:1434-1437). In fact, thedata presented herein demonstrate that the long form ObR signals throughactivation of STAT proteins—a hallmark of the signal transductionpathway mediated by the IL-6 type cytokine receptor family.

An alignment between the extracellular domains of the murine ObR andgp130 gp130 (SEQ ID NO:5) is shown in FIG. 4. Although the overall aminoacid sequence identity between these two molecules is low (24%), thecharacteristically conserved cysteine residues, the Trp-Ser-X-Trp-Sermotif (SEQ ID NO:6; amino acid residues 317-321 and 620-624 in themurine sequence shown in FIGS. 1A-1D; amino acid residues 319-323 and622-626 in the human sequence shown in FIG. 3A-3F), and conservation ofother residues within this group of proteins (reviewed in Kishimoto etat., 1994, Cell 76:253-262) is clearly evident. The amino acid sequencesof murine short form ObR and human ObR are highly homologous throughoutthe length of murine short form ObR FIGS. 5A-5B). In fact, the deducedamino acid sequence identity between the murine short form and humanclones (78%) is the same or greater than that seen when comparing themurine and human forms gp130 (Saito et at., 1992, J. Immunol.148:4066-4071), the LIF receptor (Gough et al., 1988, Proc. Natl. Acad.Sci. USA 85:2623-2627), and the G-CSF receptor (Fukanaga et al., 1990,Proc. Natl. Acad. Sci. USA 87:8702-8706). Similarly, the deduced aminoacid sequences from murine and human long forms of ObR are homologousthroughout the length of the coding region and share 75% identity FIGS.7A-7B).

Potential N-linked glycosylation sites (i.e., amino acid sequence motifN-X-S or N-X-T) are found in the ECD of both murine and human ObR. Atleast twenty potential N-linked glycosylation sites can be identified inthe murine ObR ECD sequence shown in FIGS. 1A-1D and 6A-6F (seetripeptide motifs starting at amino acid residues 23, 41, 56, 73, 81,98, 187, 206, 276, 347, 397, 433, 516, 624, 659, 670, 688, 697, 728, and750); whereas at least sixteen potential N-linked glycosylation sitescan be identified in the human ObR ECD sequence shown in FIGS. 3A-3F(see tripeptide motifs starting at amino acid residues 41, 56, 73, 98,187, 275, 345, 431, 514, 622, 657, 668, 686, 695, 698 and 726). Theextracellular domain of both the murine and human ObR is followed by apredicted transmembrane domain of 23 amino acids.

The murine cDNA shown in FIGS. 1A-1D encode a short cytoplasmic domain(34 amino acids). Amino acids 5-24 of the murine ObR cytoplasmic domain(i.e., amino acid residues 865 to 884 in FIGS. 1A-1D) show 47% identityto membrane proximal sequences of the intracellular domain of the LIFreceptor, and contain a box1 Jak interaction sequence (Narazaki et al.,1994, Proc. Natl. Acad. Sci. USA 91:2285-2289). Interestingly, the humancDNA encodes a protein with a much longer intracellular domain thanmurine short form ObR. Although the murine short form and humanintracellular domains are highly conserved up to the final five residuesof murine short form ObR, the human intracellular domain continues to alength similar to that of gp130. The nucleotide sequences of the marineshort form and human clones are also very similar throughout the codingregion of murine short form ObR, but then diverge completely near themurine short form ObR stop codon.

The short cytoplasmic domain of the murine short form cDNAs describedherein is characteristic of several class I cytokine receptorpolypeptides (reviewed in Kishimoto et al., 1994, Cell 76:253-262).However, the results reported herein demonstrate that the short form ObRdoes not activate signal transduction via the STAT pathway which ismediated by the long form ObR. In fact, the three receptors to which ObRshows the strongest homology all have long cytoplasmic domains importantin intracellular signaling. This opened the possibility that the murineshort form ObR clone isolated was chimeric or encoded a rare aberrantlyspliced form not representing the major form expressed within thechoroid plexus. To address this issue, eight murine clones were selectedthat were independently identified in the library screen, and each wasamplified (in subpools of 150 clones each) by PCR with primers made tosequences 3′ of the stop codon. Results verified that all eight clonescontained these same 3′ untranslated sequences. In addition, theC-terminus of five independently isolated clones was sequenced and allshown to have the same stop codon. Finally, reverse transcription PCRwith total RNA from choroid plexus isolated from a mouse strain(C57Bl/KsJ) different from that which the cDNA library was derived,generated an identical PCR product containing a stop codon in the samelocation. These data indicated that the isolated murine short form cloneis neither chimeric nor a rare aberrant splice event, but rather islikely to be the predominant form of this receptor in the murine choroidplexus. The data presented herein indicate that in some tissues,alternatively spliced forms of mouse ObR exist with longer intracellulardomains (the long form); i.e., the wild-type obR gene is expressed intwo forms, one mRNA transcript having an insert of about 100 nucleotidesencodes ObR having a short cytoplasmic domain, and another mRNAtranscript encodes ObR having a long cytoplasmic domain that ishomologous to the human CD.

The murine cDNA shown in FIGS. 6A-6F encode the long form ObR. Asdescribed supra, the amino acids encoding the ECD and TM of the murinelong form ObR are identical to those for the murine short form. Themurine long form cDNA, however, encodes a cytoplasmic domain (302 aminoacids) that is approximately the same length as the cytoplasmic domainencoded by the human ObR cDNA. Unlike the ObR short forms, the ObRencoded by the nucleotide sequence of the marine long form continues tobe similar to that of the human ObR throughout the cytoplasmic domain.

The data presented herein also indicate that db is a mutant of the longform murine obR gene. The db mutant expresses an aberrantly splicedtranscript containing an insert of about 106 nucleotides in the portionof the mRNA encoding the CD. Although the transcript is long, theinserted sequence produces a mutation that results in a transcript thatencodes a truncated ObR protein that is identical to the short forms ofObR and therefore, lacks most of the CD. The data shown hereindemonstrate that, unlike the long form ObR, the short form ObR, i.e.,the form of the receptor associated with the obese phenotype in db/dbmice, does not transduce signal mediated by the STAT pathway. Therefore,it appears that the signalling-competant long form ObR is activelyinvolved in body weight regulation and maintenance.

In sum, messenger RNA for several major ObR forms have been identified.The predominant ObR mRNA found in most tissues encodes a transmembraneprotein with a short cytoplasmic domain of 34 amino acid residuesreferred to as the short form. In hypothalamus, an obR mRNA exists whichencodes a protein with an identical extracellular domain as the shortform, but with a 302 residue-long cytoplasmic domain, referred to as thelong form. The db mutation leads to the production of an aberrant spliceproduct of long form transcript, resulting in a protein with truncatedcytoplasmic domain. Interestingly, the mRNA for the long form of ObR inthe db/db mice encodes a protein with an identical structure to thenaturally occurring short form. The loss of this carboxyterminal regionis proposed to render the ObR inactive and is predicted to generate theobese phenotype in db/db mice.

Sequence information indicated that ObR might exert a signaling actionsimilar to that of G-CSFR, LIFR, and gp130 (Stahl & Yancopoulos, 1993,Cell 74:587-590; Kishimoto, et al., 1995, Blood 86:1243-1254). Signalingby these receptors entails, among others, the activation ofreceptor-associated kinases of the Janus kinase family which contributeto the phosphorylation and activation of the DNA binding activity ofSTAT1, STAT3 and STAT5 (Ihle, 1995, Nature 377:591-594; Kishimoto, etal., 1995, Blood 86:1243-1254). This process, in turn, has beencorrelated with induced transcription of genes that contain bindingsites for the STAT proteins such as the hepatic genes encoding acutephase plasma proteins (Lai et al., 1995, J. Biol. Chem.270:23254-23257). To address whether the cloned ObR isoforms are indeedsignaling receptor molecules, ObR was introduced into established tissueculture cell lines and the cell response to OB treatment was comparedwith that mediated by the structurally-related IL-6-type cytokinereceptors. The results presented in the example infra demonstrate thatthe long form ObR is a signal-transducing molecule. In particular, theresults show that the long form ObR shares functional specificity withIL-6-type cytokine receptors. The results also show that the short formObR does not signal via the STAT pathway transduced by the ObR longform. Thus, it appears that the long form ObR, but not the short form,is involved in maintenance of body weight.

The ObR amino acid sequences of the invention include the amino acidsequence shown in FIGS. 1A-1D (SEQ ID NO:2), FIGS. 3A-3F (SEQ ID NO:4)or FIGS. 6A-6F, or the amino acid sequence encoded by cDNA clonefamj5312 as deposited with the ATCC, or encoded by cDNA clone fahj5312das deposited with the ATCC, or encoded by the human genomic cloneh-obR-p87, as deposited with the ATCC. Further, ObRs of other speciesare encompassed by the invention. In fact, any ObR protein encoded bythe obR nucleotide sequences described in Section 5.1, above, are withinthe scope of the invention.

The invention also encompasses proteins that are functionally equivalentto the ObR encoded by the nucleotide sequences described in Section 5.1,as judged by any of a number of criteria, including but not limited tothe ability to bind Ob, the binding affinity for Ob, the resultingbiological effect of Ob binding, for example, signal transduction, achange in cellular metabolism (e.g., ion flux, tyrosine phosphorylation)or change in phenotype when the ObR equivalent is present in anappropriate cell type (such as the amelioration, prevention or delay ofthe obese phenotype, i.e., the db or ob phenotype), or weight loss. Suchfunctionally equivalent ObR proteins include but are not limited toadditions or substitutions of amino acid residues within the amino acidsequence encoded by the obR nucleotide sequences described, above, inSection 5.1, but which result in a silent change, thus producing afunctionally equivalent gene product. Amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues involved. For example, nonpolar (hydrophobic) amino acidsinclude alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, and methionine; polar neutral amino acids include glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;positively charged (basic) amino acids include arginine, lysine, andhistidine; and negatively charged (acidic) amino acids include asparticacid and glutamic acid.

While random mutations can be made to obR DNA (using random mutagenesistechniques well known to those skilled in the art) and the resultingmutant ObRs tested for activity, site-directed mutations of the obRcoding sequence can be engineered (using site-directed mutagenesistechniques well known to those skilled in the art) to generate mutantObRs with increased function, for example, higher binding affinity forOb, and/or greater signalling capacity; or decreased function, forexample, lower binding affinity for Ob, and/or decreased signaltransduction capacity.

For example, the alignment of mouse short form ObR FIGS. 1A-1D) and thehuman ObR homolog FIGS. 3A-3F) is shown in FIGS. 5A-5B in whichidentical amino acid residues are indicated by a star. Mutant ObRs canbe engineered so that regions of identity (indicated by stars in FIGS.5A-5B) are maintained, whereas the variable residues (unstarred in FIGS.5A-5B) are altered, for example, by deletion or insertion of an aminoacid residue(s) or by substitution of one or more different amino acidresidues. Conservative alterations at the variable positions can beengineered in order to produce a mutant ObR that retains function; forexample, Ob binding affinity or signal transduction capability or both.Non-conservative changes can be engineered at these variable positionsto alter function, for example, Ob binding affinity or signaltransduction capability, or both. Alternatively, where alteration offunction is desired, deletion or non-conservative alterations of theconserved regions (i.e., identical amino acids indicated by stars inFIGS. 5A-5B) can be engineered. For example, deletion ornon-conservative alterations (substitutions or insertions) of the CD,for example, amino acid residues 861-894 (FIGS. 1A-1D) of murine ObR, oramino acid residues 863-1165 (FIGS. 3A-3F) of human ObR, or portions ofthe CD, for example, amino acid residues 861-884 (FIGS. 1A-1D) of murineObR, or amino acid residues 863-886 (FIGS. 3A-3F) of human ObR (the box1 Jak interaction domain) can be engineered to produce a mutant ObR thatbinds Ob but is signalling-incompetent. Non-conservative alterations tothe starred residues in the ECD shown in FIGS. 5A-5B can be engineeredto produce mutant ObRs with altered binding affinity for Ob. The samemutation strategy can also be used to design mutant ObRs based on thealignment of murine long ObR form and the human ObR homolog shown inFIGS. 7A-7B in which identical amino acid residues are indicated by adouble asterisk.

FIG. 4 shows the alignment of the ECD of murine ObR with human gp130, inwhich identical residues are indicated in black, and conservativechanges are indicated in grey. Presumably, regions of identity andconservation are important for maintaining tertiary structure of theECD, whereas the variable regions may contribute to specificity of eachreceptor for its ligand. Therefore, ObR mutants with altered bindingaffinity for Ob may be engineered by altering the variable regions shownin FIG. 4. Such ObR mutants can be designed so as to preserve the ObRamino acid sequences that are boxed in FIG. 4 (both black and greyboxes) or to contain one or more conservative substitutions derived fromthe gp130 sequence shown in the grey boxes of FIG. 4.

Other mutations to the obR coding sequence can be made to generate ObRsthat are better suited for expression, scale up, etc. in the host cellschosen. For example, cysteine residues can be deleted or substitutedwith another amino acid in order to eliminate disulfide bridges;N-linked glycosylation sites can be altered or eliminated to achieve,for example, expression of a homogeneous product that is more easilyrecovered and purified from yeast hosts which are known tohyperglycosylate N-linked sites. To this end, a variety of amino acidsubstitutions at one or both of the first or third amino acid positionsof any one or more of the glycosylation recognition sequences whichoccur in the ECD (N-X-S or N-X-T), and/or an amino acid deletion at thesecond position of any one or more such recognition sequences in the ECDwill prevent glycosylation of the ObR at the modified tripeptidesequence. (See, e.g., Miyajima et al., 1986, EMBO J. 5:1193-1197).

Peptides corresponding to one or more domains of the ObR (e.g., ECD, TM,or CD), truncated or deleted ObRs (e.g., ObR in which the TM and/or CDis deleted) as well as fusion proteins in which the full length ObR, anObR peptide or truncated ObR is fused to an unrelated protein are alsowithin the scope of the invention and can be designed on the basis ofthe obR nucleotide and ObR amino acid sequences disclosed in thisSection and in Section 5.1, above. Such fusion proteins include but arenot limited to IgFc fusions which stabilize the ObR protein or peptideand prolong half-life in vivo; or fusions to any amino acid sequencethat allows the fusion protein to be anchored to the cell membrane,allowing the ECD to be exhibited on the cell surface; or fusions to anenzyme, fluorescent protein, or luminescent protein which provide amarker function.

While the ObR polypeptides and peptides can be chemically synthesized(e.g., see Creighton, 1983, Proteins: Structures and MolecularPrinciples, W. H. Freeman & Co., N.Y.), large polypeptides derived fromthe ObR and the full length ObR itself may advantageously be produced byrecombinant DNA technology using techniques well known in the art forexpressing nucleic acid containing obR gene sequences and/or codingsequences. Such methods can be used to construct expression vectorscontaining the obR nucleotide sequences described in Section 5.1 andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. See, forexample, the techniques described in Sambrook et al., 1989, supra, andAusubel et al., 1989, supra. Alternatively, RNA capable of encoding obRnucleotide sequences may be chemically synthesized using, for example,synthesizers. See, for example, the techniques described inOligonucleotide Synthesis, 1984, Gait, M. J. ed., IRL Press, Oxford,which is incorporated by reference herein in its entirety.

A variety of host-expression vector systems may be utilized to expressthe obR nucleotide sequences of the invention. Where the ObR peptide orpolypeptide is a soluble derivative (e.g., ObR peptides corresponding tothe ECD; truncated or deleted ObR in which the TM and/or CD are deleted)the peptide or polypeptide can be recovered from the culture, i.e., fromthe host cell in cases where the ObR peptide or polypeptide is notsecreted, and from the culture media in cases where the ObR peptide orpolypeptide is secreted by the cells. However, the expression systemsalso encompass engineered host cells that express the ObR or functionalequivalents in situ, i.e., anchored in the cell membrane. Purificationor enrichment of the ObR from such expression systems can beaccomplished using appropriate detergents and lipid micelles and methodswell known to those skilled in the art. However, such engineered hostcells themselves may be used in situations where it is important notonly to retain the structural and functional characteristics of the ObR,but to assess biological activity, e.g., in drug screening assays.

The expression systems that may be used for purposes of the inventioninclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing obR nucleotidesequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing the obR nucleotidesequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing the obR sequences;plant cell systems infected with recombinant virus expression vectors(e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) ortransformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing obR nucleotide sequences; or mammalian cell systems(e.g., COS, CHO, BHK, 293, 3T3) harboring recombinant expressionconstructs containing promoters derived from the genome of mammaliancells (e.g., metallothionein promoter) or from mammalian viruses (e.g.,the adenovirus late promoter; the vaccinia virus 7.5K promoter).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the obR geneproduct being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of ObR protein or for raising antibodies to the ObRprotein, for example, vectors which direct the expression of high levelsof fusion protein products that are readily purified may be desirable.Such vectors include, but are not limited to, the E. coli expressionvector pUR278 (Ruther et al., 1983, EMBO J. 2:1791), in which the obRcoding sequence may be ligated individually into the vector in framewith the lacZ coding region so that a fusion protein is produced; pINvectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109; VanHeeke & Schuster, 1989, J. Biol. Chem. 264:5503-5509); and the like.pGEX vectors may also be used to express foreign polypeptides as fusionproteins with glutathione S-transferase (GST). In general, such fusionproteins are soluble and can easily be purified from lysed cells byadsorption to glutathione-agarose beads followed by elution in thepresence of free glutathione. The PGEX vectors are designed to includethrombin or factor Xa protease cleavage sites so that the cloned targetgene product can be released from the GST moiety.

In an insect system, Autographa californica, nuclear polyhidrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The obR gene coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter). Successful insertion of obR genecoding sequence will result in inactivation of the polyhedrin gene andproduction of non-occluded recombinant virus (i.e., virus lacking theproteinaceous coat coded for by the polyhedrin gene). These recombinantviruses are then used to infect Spodoptera frugiperda cells in which theinserted gene is expressed. (e.g., see Smith et al., 1983, J. Virol.46:584; Smith, U.S. Pat. No. 4,215,051).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the obR nucleotide sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the obR gene product in infected hosts. (E.g., See Logan &Shenk, 1984, Proc. Natl. Acad. Sci. USA 81:3655-3659). Specificinitiation signals may also be required for efficient translation ofinserted obR nucleotide sequences. These signals include the ATGinitiation codon and adjacent sequences. In cases where an entire obRgene or cDNA, including its own initiation codon and adjacent sequences,is inserted into the appropriate expression vector, no additionaltranslational control signals may be needed. However, in cases whereonly a portion of the obR coding sequence is inserted, exogenoustranslational control signals, including, perhaps, the ATG initiationcodon, must be provided. Furthermore, the initiation codon must be inphase with the reading frame of the desired coding sequence to ensuretranslation of the entire insert. These exogenous translational controlsignals and initiation codons can be of a variety of origins, bothnatural and synthetic. The efficiency of expression may be enhanced bythe inclusion of appropriate transcription enhancer elements,transcription terminators, etc. (See Bittner et al., 1987, Methods inEnzymol. 153:516-544).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK,293, 3T3, WI38, and in particular, choroid plexus cell lines.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe obR sequences described above may be engineered. Rather than usingexpression vectors which contain viral origins of replication, hostcells can be transformed with DNA controlled by appropriate expressioncontrol elements (e.g., promoter, enhancer sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the obR geneproduct. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the obR gene product.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler, et al., 1977, Cell11:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adeninephosphoribosyltransferase (Lowy, et al., 1980, Cell 22:817) genes can beemployed in tk⁻, hgprt⁻ or aprt⁻ cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., 1980, Proc. Natl. Acad. Sci. USA 77:3567; O'Hare, et al., 1981,Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA78:2072); neo, which confers resistance to the aminoglycoside G-418(Colberre-Garapin, et al., 1981, J. Mol. Biol. 150:1); and hygro, whichconfers resistance to hygromycin (Santerre, et al., 1984, Gene 30:147).

Alternatively, any fusion protein may be readily purified by utilizingan antibody specific for the fusion protein being expressed. Forexample, a system described by Janknecht et al. allows for the readypurification of non-denatured fusion proteins expressed in human celllines (Janknecht, et al., 1991, Proc. Natl. Acad. Sci. USA88:8972-8976). In this system, the gene of interest is subcloned into avaccinia recombination plasmid such that the gene's open reading frameis translationally fused to an amino-terminal tag consisting of sixhistidine residues. Extracts from cells infected with recombinantvaccinia virus are loaded onto Ni²⁺.nitriloacetic acid-agarose columnsand histidine-tagged proteins are selectively eluted withimidazole-containing buffers.

The obR gene products can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates,for example, baboons, monkeys, and chimpanzees may be used to generateobR transgenic animals.

Any technique known in the art may be used to introduce the obRtransgene into animals to produce the founder lines of transgenicanimals. Such techniques include, but are not limited to pronuclearmicroinjection (Hoppe, P. C. and Wagner, T. E., 1989, U.S. Pat. No.4,873,191); retrovirus mediated gene transfer into germ lines (Van derPutten et al., 1985, Proc. Natl. Acad. Sci. USA 82:6148-6152); genetargeting in embryonic stem cells (Thompson et al., 1989, Cell56:313-321); electroporation of embryos (Lo, 1983, Mol. Cell. Biol.3:1803-1814); and sperm-mediated gene transfer (Lavitrano et al., 1989,Cell 57:717-723); etc. For a review of such techniques, see Gordon,1989, Transgenic Animals, Intl. Rev. Cytol. 115:171-229, which isincorporated by reference herein in its entirety.

The present invention provides for transgenic animals that carry the obRtransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals. Thetransgene may be integrated as a single transgene or in concatamers,e.g., head-to-head tandems or head-to-tail tandems. The transgene mayalso be selectively introduced into and activated in a particular celltype by following, for example, the teaching of Lasko et al. (Lasko, M.et al., 1992, Proc. Natl. Acad. Sci. USA 89:6232-6236). The regulatorysequences required for such a cell-type specific activation will dependupon the particular cell type of interest, and will be apparent to thoseof skill in the art. When it is desired that the obR gene transgene beintegrated into the chromosomal site of the endogenous obR gene, genetargeting is preferred. Briefly, when such a technique is to beutilized, vectors containing some nucleotide sequences homologous to theendogenous obR gene are designed for the purpose of integrating, viahomologous recombination with chromosomal sequences, into and disruptingthe function of the nucleotide sequence of the endogenous obR gene. Thetransgene may also be selectively introduced into a particular celltype, thus inactivating the endogenous obR gene in only that cell type,by following, for example, the teaching of Gu et al. (Gu, et al., 1994,Science 265:103-106). The regulatory sequences required for such acell-type specific inactivation will depend upon the particular celltype of interest, and will be apparent to those of skill in the art.

Once transgenic animals have been generated, the expression of therecombinant obR gene may be assayed utilizing standard techniques.Initial screening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to assay whether integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include but are not limited to Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and RT-PCR. Samples of obR gene-expressing tissue, may also beevaluated immunocytochemically using antibodies specific for the obRtransgene product.

5.3. Antibodies to ObR Proteins

Antibodies that specifically recognize one or more epitopes of ObR, orepitopes of conserved variants of ObR, or peptide fragments of the ObRare also encompassed by the invention. Such antibodies include but arenot limited to polyclonal antibodies, monoclonal antibodies (mAbs),humanized or chimeric antibodies, single chain antibodies, Fabfragments, F(ab′)₂ fragments, fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, and epitope-bindingfragments of any of the above.

The antibodies of the invention may be used, for example, in thedetection of the ObR in a biological sample and may, therefore, beutilized as part of a diagnostic or prognostic technique wherebypatients may be tested for abnormal amounts of ObR. Such antibodies mayalso be utilized in conjunction with, for example, compound screeningschemes, as described, below, in Section 5.5, for the evaluation of theeffect of test compounds on expression and/or activity of the obR geneproduct. Additionally, such antibodies can be used in conjunction withthe gene therapy techniques described, below, in Section 5.6, to, forexample, evaluate the normal and/or engineered ObR-expressing cellsprior to their introduction into the patient. Such antibodies mayadditionally be used as a method for the inhibition of abnormal ObRactivity. Thus, such antibodies may, therefore, be utilized as part ofweight disorder treatment methods.

For the production of antibodies, various host animals may be immunizedby injection with the ObR, an ObR peptide (e.g., one corresponding the afunctional domain of the receptor, such as ECD, TM or CD), truncated ObRpolypeptides (ObR in which one or more domains, e.g., the TM or CD, hasbeen deleted), functional equivalents of the ObR or mutants of the ObR.Such host animals may include but are not limited to rabbits, mice, andrats, to name but a few. Various adjuvants may be used to increase theimmunological response, depending on the host species, including but notlimited to Freund's (complete and incomplete), mineral gels such asaluminum hydroxide, surface active substances such as lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpethemocyanin, dinitrophenol, and potentially useful human adjuvants suchas BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Polyclonalantibodies are heterogeneous populations of antibody molecules derivedfrom the sera of the immunized animals.

Monoclonal antibodies, which are homogeneous populations of antibodiesto a particular antigen, may be obtained by any technique which providesfor the production of antibody molecules by continuous cell lines inculture. These include, but are not limited to, the hybridoma techniqueof Kohler and Milstein (1975, Nature 256:495-497; and U.S. Pat. No.4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983,Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985,Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp.77-96). Such antibodies may be of any immunoglobulin class includingIgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridomaproducing the mAb of this invention may be cultivated in vitro or invivo. Production of high titers of mAbs in vivo makes this the presentlypreferred method of production.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA81:6851-6855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al.,1985, Nature 314:452-454) by splicing the genes from a mouse antibodymolecule of appropriate antigen specificity together with genes from ahuman antibody molecule of appropriate biological activity can be used.A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a murine mAb and a human immunoglobulin constantregion.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242:423-426;Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Wardet al., 1989, Nature 334:544-546) can be adapted to produce single chainantibodies against obR gene products. Single chain antibodies are formedby linking the heavy and light chain fragments of the Fv region via anamino acid bridge, resulting in a single chain polypeptide.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, such fragments include but are notlimited to: the F(ab′)₂ fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)₂ fragments.Alternatively, Fab expression libraries may be constructed (Huse et al.,1989, Science 246:1275-1281) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.

Antibodies to the ObR can, in turn, be utilized to generateanti-idiotype antibodies that “mimic” the ObR, using techniques wellknown to those skilled in the art. (See, e.g., Greenspan & Bona, 1993,FASEB J. 7 (5):437-444; and Nissinoff, 1991, J. Immunol. 147(8):2429-2438). For example, antibodies which bind to the ObR ECD andcompetitively inhibit the binding of Ob to the ObR can be used togenerate anti-idiotypes that “mimic” the ECD and, therefore, bind andneutralize Ob. Such neutralizing anti-idiotypes or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens to neutralize Ob andpromote weight gain.

5.4. Diagnosis of Body Weight Disorder Abnormalities

A variety of methods can be employed for the diagnostic and prognosticevaluation of body weight disorders, including obesity, cachexia andanorexia, and for the identification of subjects having a predispositionto such disorders.

Such methods may, for example, utilize reagents such as the obRnucleotide sequences described in Section 5.1, and ObR antibodies, asdescribed, in Section 5.3. Specifically, such reagents may be used, forexample, for: (1) the detection of the presence of obR gene mutations,or the detection of either over- or under-expression of obR mRNArelative to the non-body weight disorder state; (2) the detection ofeither an over- or an under-abundance of obR gene product relative tothe non-body weight disorder state; and (3) the detection ofperturbations or abnormalities in the signal transduction pathwaymediated by ObR.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one specific obRnucleotide sequence or ObR antibody reagent described herein, which maybe conveniently used, e.g., in clinical settings, to diagnose patientsexhibiting body weight disorder abnormalities.

For the detection of obR mutations, any nucleated cell can be used as astarting source for genomic nucleic acid. For the detection of obR geneexpression or obR gene products, any cell type or tissue in which theobR gene is expressed, such as, for example, choroid plexus cells, maybe utilized.

Nucleic acid-based detection techniques are described, below, in Section5.4.1. Peptide detection techniques are described, below, in Section5.4.2.

5.4.1. Detection of the obR Gene and Transcripts

Mutations within the obR gene can be detected by utilizing a number oftechniques. Nucleic acid from any nucleated cell can be used as thestarting point for such assay techniques, and may be isolated accordingto standard nucleic acid preparation procedures which are well known tothose of skill in the art.

DNA may be used in hybridization or amplification assays of biologicalsamples to detect abnormalities involving obR gene structure, includingpoint mutations, insertions, deletions and chromosomal rearrangements.Such assays may include, but are not limited to, Southern analyses,single stranded conformational polymorphism analyses (SSCP), and PCRanalyses.

Such diagnostic methods for the detection of obR gene-specific mutationscan involve for example, contacting and incubating nucleic acidsincluding recombinant DNA molecules, cloned genes or degenerate variantsthereof, obtained from a sample, for example, derived from a patientsample or other appropriate cellular source, with one or more labelednucleic acid reagents including recombinant DNA molecules, cloned genesor degenerate variants thereof, as described in Section 5.1, underconditions favorable for the specific annealing of these reagents totheir complementary sequences within the obR gene. Preferably, thelengths of these nucleic acid reagents are at least 15 to 30nucleotides. After incubation, all non-annealed nucleic acids areremoved from the nucleic acid:obR molecule hybrid. The presence ofnucleic acids which have hybridized, if any such molecules exist, isthen detected. Using such a detection scheme, the nucleic acid from thecell type or tissue of interest can be immobilized, for example, to asolid support such as a membrane, or a plastic surface such as that on amicrotiter plate or polystyrene beads. In this case, after incubation,non-annealed, labeled nucleic acid reagents of the type described inSection 5.1 are easily removed. Detection of the remaining, annealed,labeled obR nucleic acid reagents is accomplished using standardtechniques well-known to those in the art. The obR gene sequences towhich the nucleic acid reagents have annealed can be compared to theannealing pattern expected from a normal obR gene sequence in order todetermine whether an obR gene mutation is present.

Alternative diagnostic methods for the detection of obR gene specificnucleic acid molecules, in patient samples or other appropriate cellsources, may involve their amplification, for example, by PCR (theexperimental embodiment set forth in Mullis, K. B., 1987, U.S. Pat. No.4,683,202), followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. The resultingamplified sequences can be compared to those which would be expected ifthe nucleic acid-being amplified contained only normal copies of the obRgene in order to determine whether an obR gene mutation exists.

Additionally, well-known genotyping techniques can be performed toidentify individuals carrying obR gene mutations. Such techniquesinclude, for example, the use of restriction fragment lengthpolymorphisms (RFLPs), which involve sequence variations in one of therecognition sites for the specific restriction enzyme used.

Additionally, improved methods for analyzing DNA polymorphisms, whichcan be utilized for the identification of obR gene mutations, have beendescribed. These methods capitalize on the presence of variable numbersof short, tandemly repeated DNA sequences between the restriction enzymesites. For example, Weber (U.S. Pat. No. 5,075,217, which isincorporated herein by reference in its entirety) describes a DNA markerbased on length polymorphisms in blocks of (dC-dA)_(n)-(dG-dT)_(n) shorttandem repeats. The average separation of (dC-dA)_(n)-(dG-dT)_(n) blocksis estimated to be 30,000-60,000 bp. Markers that are so closely spacedexhibit a high frequency co-inheritance, and are extremely useful in theidentification of genetic mutations, such as, for example, mutationswithin the obR gene, and the diagnosis of diseases and disorders relatedto obR mutations.

Also, Caskey et al. (U.S. Pat. No. 5,364,759, which is incorporatedherein by reference in its entirety) describe a DNA profiling assay fordetecting short tri- and tetra-nucleotide repeat sequences. The processincludes extracting the DNA of interest, such as the obR gene,amplifying the extracted DNA, and labelling the repeat sequences to forma genotypic map of the individual's DNA.

The level of obR gene expression can also be assayed by detecting andmeasuring obR transcription. For example, RNA from a cell type or tissueknown, or suspected, to express the obR gene, such as brain, especiallychoroid plexus cells, can be isolated and tested utilizing hybridizationor PCR techniques such as those described above. The isolated cells canbe obtained from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cells tobe used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of theobR gene. Such analyses can reveal both quantitative and qualitativeaspects of the expression pattern of the obR gene, including activationor inactivation of obR gene expression.

In one embodiment of such a detection scheme, cDNAs are synthesized fromthe RNAs of interest (e.g., by reverse transcription of the RNA moleculeinto cDNA). A sequence within the cDNA is then used as the template fora nucleic acid amplification reaction, such as a PCR amplificationreaction, or the like. The nucleic acid reagents used as synthesisinitiation reagents (e.g., primers) in the reverse transcription andnucleic acid amplification steps of this method are chosen from amongthe obR nucleic acid reagents described in Section 5.1. The preferredlengths of such nucleic acid reagents are at least 9-30 nucleotides. Fordetection of the amplified product, the nucleic acid amplification maybe performed using radioactively or non-radioactively labelednucleotides. Alternatively, enough amplified product can be made suchthat the product can be visualized by standard ethidium bromide stainingor by utilizing any other suitable nucleic acid staining method.

Additionally, it is possible to perform such obR gene expression assaysin situ, i.e., directly upon tissue sections (fixed and/or frozen) ofpatient tissue obtained from biopsies or resections, such that nonucleic acid purification is necessary. Nucleic acid reagents such asthose described in Section 5.1 may be used as probes and/or primers forsuch in situ procedures (See, e.g., Nuovo, G. J., 1992, PCR In SituHybridization: Protocols And Applications, Raven Press, NY).

Alternatively, if a sufficient quantity of the appropriate cells can beobtained, standard Northern analysis can be performed to determine thelevel of mRNA expression of the obR gene.

5.4.2. Detection of the obR Gene Products

Antibodies directed against wild type or mutant obR gene products orconserved variants or peptide fragments thereof, which are discussed,above, in Section 5.3, can also be used as body weight disorderdiagnostics and prognostics, as described herein. Such diagnosticmethods, can be used to detect abnormalities in the level of obR geneexpression, or abnormalities in the structure and/or temporal, tissue,cellular, or subcellular location of the ObR, and may be performed invivo or in vitro, such as, for example, on biopsy tissue.

For example, antibodies directed to epitopes of the ObR ECD can be usedin vivo to detect the pattern and level of expression of the ObR in thebody. Such antibodies can be labeled, for example, with a radio-opaqueor other appropriate compound and injected into a subject in order tovisualize binding to the ObR expressed in the body using methods such asX-rays, CAT-scans, or MRI. Labeled antibody fragments, for example, theFab or single chain antibody comprising the smallest portion of theantigen binding region, are preferred for this purpose to promotecrossing the blood-brain barrier and permit labeling ObRs expressed inthe choroid plexus.

Additionally, any ObR fusion protein or ObR conjugated protein whosepresence can be detected, can be administered. For example, ObR fusionor conjugated proteins labeled with a radio-opaque or other appropriatecompound can be administered and visualized in vivo, as discussed, abovefor labeled antibodies. Further such Ob fusion proteins as AP-Ob onOb-Ap fusion proteins can be utilized for in vitro diagnosticprocedures.

Alternatively, immunoassays or fusion protein detection assays, asdescribed above, can be utilized on biopsy and autopsy samples in vitroto permit assessment of the expression pattern of the ObR. Such assaysare not confined to the use of antibodies that define the ObR ECD, butcan include the use of antibodies directed to epitopes of any of thedomains of the ObR, for example, the ECD, the TM and/or CD. The use ofeach or all of these labeled antibodies will yield useful informationregarding translation and intracellular transport of the ObR to the cellsurface, and can identify defects in processing.

The tissue or cell type to be analyzed will generally include thosewhich are known, or suspected, to express the obR gene, such as, forexample, choroid plexus cells. The protein isolation methods employedherein can, for example, be such as those described in Harlow and Lane(Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.), which isincorporated herein by reference in its entirety. The isolated cells canbe derived from cell culture or from a patient. The analysis of cellstaken from culture may be a necessary step in the assessment of cellsthat could be used as part of a cell-based gene therapy technique or,alternatively, to test the effect of compounds on the expression of theobR gene.

For example, antibodies, or fragments of antibodies, such as thosedescribed, above, in Section 5.3, useful in the present invention may beused to quantitatively or qualitatively detect the presence of obR geneproducts or conserved variants or peptide fragments thereof. This can beaccomplished, for example, by immunofluorescence techniques employing afluorescently labeled antibody (see below, this Section) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Suchtechniques are especially preferred if such obR gene products areexpressed on the cell surface.

The antibodies (or fragments thereof) or Ob fusion or conjugatedproteins useful in the present invention can, additionally, be employedhistologically, as in immunofluorescence, immunoelectron microscopy ornon-immuno assays, for in situ detection of obR gene products orconserved variants or peptide fragments thereof, or for Ob binding (inthe case of labeled Ob fusion protein).

In situ detection may be accomplished by removing a histologicalspecimen from a patient, and applying thereto a labeled antibody orfusion protein of the present invention. The antibody (or fragment) orfusion protein is preferably applied by overlaying the labeled antibody(or fragment) onto a biological sample. Through the use of such aprocedure, it is possible to determine not only the presence of the obRgene product, or conserved variants or peptide fragments, or Ob binding,but also its distribution in the examined tissue. Using the presentinvention, those of ordinary skill will readily perceive that any of awide variety of histological methods (such as staining procedures) canbe modified in order to achieve such in situ detection.

Immunoassays and non-immunoassays for obR gene products or conservedvariants or peptide fragments thereof will typically comprise incubatinga sample, such as a biological fluid, a tissue extract, freshlyharvested cells, or lysates of cells which have been incubated in cellculture, in the presence of a detectably labeled antibody capable ofidentifying obR gene products or conserved variants or peptide fragmentsthereof, and detecting the bound antibody by any of a number oftechniques well-known in the art.

The biological sample may be brought in contact with and immobilizedonto a solid phase support or carrier such as nitrocellulose, or othersolid support which is capable of immobilizing cells, cell particles, orsoluble proteins. The support may then be washed with suitable buffersfollowed by treatment with the detectably labeled ObR antibody or Obfusion protein. The solid phase support may then be washed with thebuffer a second time to remove unbound antibody or fusion protein. Theamount of bound label on solid support may then be detected byconventional means.

By “solid phase support or carrier” is intended any support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

The binding activity of a given lot of ObR antibody or Ob fusion proteinmay be determined according to well known methods. Those skilled in theart will be able to determine operative and optimal assay conditions foreach determination by employing routine experimentation.

With respect to antibodies, one of the ways in which the ObR antibodycan be detectably labeled is by linking the same to an enzyme and use inan enzyme immunoassay (EIA) (Voller, A., The Enzyme Linked ImmunosorbentAssay (ELISA), 1978, Diagnostic Horizons 2:1-7, MicrobiologicalAssociates Quarterly Publication, Walkersville, Md.); Voller, A. et al.,1978, J. Clin. Pathol. 31:507-520; Butler, J. E., 1981, Meth. Enzymol.73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, BocaRaton, Fla.; Ishikawa, E. et al., (eds.), 1981, Enzyme Immunoassay,Kgaku Shoin, Tokyo). The enzyme which is bound to the antibody willreact with an appropriate substrate, preferably a chromogenic substrate,in such a manner as to produce a chemical moiety that can be detected,for example, by spectrophotometric, fluorimetric or by visual means.Enzymes that can be used to detectably label the antibody include, butare not limited to, malate dehydrogenase, staphylococcal nuclease,delta-5-steroid isomerase, yeast alcohol dehydrogenase,alpha-glycerophosphate, dehydrogenase, triose phosphate isomerase,horseradish peroxidase, alkaline phosphatase, asparaginase, glucoseoxidase, beta-galactosidase, ribonuclease, urease, catalase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. The detection can be accomplished by calorimetricmethods that employ a chromogenic substrate for the enzyme. Detectioncan also be accomplished by visual comparison of the extent of enzymaticreaction of a substrate in comparison with similarly prepared standards.

Detection can also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect ObR through the use of aradioimmunoassay (RIA) (see, for example, Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand AssayTechniques, The Endocrine Society, March, 1986, which is incorporated byreference herein). The radioactive isotope can be detected by such meansas the use of a gamma counter or a scintillation counter or byautoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems, in which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase, and aequorin.

5.5. Screening Assays for Compounds that Modulate ObR Expression orActivity

The following assays are designed to identify compounds that interactwith (e.g., bind to) ObR (including, but not limited to the ECD or CD ofObR), compounds that interact with (e.g., bind to) intracellularproteins that interact with ObR (including, but not limited to, the TMand CD of ObR), compounds that interfere with the interaction of ObRwith transmembrane or intracellular proteins involved in ObR-mediatedsignal transduction, and to compounds that modulate the activity of anobR gene (i.e., modulate the level of obR gene expression) or modulatethe level of ObR. Assays can additionally be utilized which identifycompounds that bind to obR gene regulatory sequences (e.g., promotersequences) and which may modulate obR gene expression. See e.g., Platt,J. Biol. Chem. 269:28558-28562, 1994, which is incorporated herein byreference in its entirety.

Compounds that can be screened in accordance with the invention include,but are not limited to peptides, antibodies and fragments thereof, andother organic compounds (e.g., peptidomimetics) that bind to the ECD ofthe ObR and either mimic the activity triggered by the natural ligand(i.e., agonists) or inhibit the activity triggered by the natural ligand(i.e., antagonists); as well as peptides, antibodies or fragmentsthereof, and other organic compounds that mimic the ECD of the ObR (or aportion thereof) and bind to and “neutralize” natural ligand.

Such compounds can include, but are not limited to, peptides such as,for example, soluble peptides, including but not limited to members ofrandom peptide libraries; (see, e.g., Lam, et al., Nature 354:82-84,1991; Houghten, et al., Nature 354:84-86, 1991), and combinatorialchemistry-derived molecular library made of D- and/or L—configurationamino acids, phosphopeptides (including, but not limited to, members ofrandom or partially degenerate, directed phosphopeptide libraries; see,e.g., Songyang, Z. et al., 1993, Cell 72:767-778), antibodies(including, but not limited to, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments, and epitope-binding fragmentsthereof), and small organic or inorganic molecules.

Other compounds that can be screened in accordance with the inventioninclude but are not limited to small organic molecules that are able tocross the blood-brain barrier, gain entry into an appropriate cell(e.g., a cell in the choroid plexus or in the hypothalamus) and affectthe expression of the obR gene or some other gene involved in the ObRsignal transduction pathway (e.g., by interacting with the regulatoryregion or transcription factors involved in gene expression); or suchcompounds that affect the activity of the ObR (e.g., by inhibiting orenhancing the enzymatic activity of the CD) or the activity of someother intracellular factor involved in the ObR signal transductionpathway, such as, for example, gp130.

Computer modelling and searching technologies permit identification ofcompounds, or the improvement of already identified compounds, that canmodulate ObR expression or activity. Having identified such a compoundor composition, the active sites or regions are identified. Such activesites might typically be ligand binding sites, such as the interactiondomains of Ob with ObR itself. The active site can be identified usingmethods known in the art including, for example, from the amino acidsequences of peptides, from the nucleotide sequences of nucleic acids,or from study of complexes of the relevant compound or composition withits natural ligand. In the latter case, chemical or X-raycrystallographic methods can be used to find the active site by findingwhere on the factor the complexed ligand is found.

Next, the three dimensional geometric structure of the active site isdetermined. This can be done by known methods, including X-raycrystallography, which can determine a complete molecular structure. Onthe other hand, solid or liquid phase NMR can be used to determinecertain intra-molecular distances. Any other experimental method ofstructure determination can be used to obtain partial or completegeometric structures. The geometric structures can be measured with acomplexed ligand, natural or artificial, which may increase the accuracyof the active site structure determined.

If an incomplete or insufficiently accurate structure is determined, themethods of computer based numerical modelling can be used to completethe structure or improve its accuracy. Any recognized modelling methodmay be used, including parameterized models specific to particularbiopolymers such as proteins or nucleic acids, molecular dynamics modelsbased on computing molecular motions, statistical mechanics models basedon thermal ensembles, or combined models. For most types of models,standard molecular force fields, representing the forces betweenconstituent atoms and groups, are necessary, and can be selected fromforce fields known in physical chemistry. The incomplete or lessaccurate experimental structures can serve as constraints on thecomplete and more accurate structures computed by these modelingmethods.

Finally, having determined the structure of the active site, eitherexperimentally, by modeling, or by a combination, candidate modulatingcompounds can be identified by searching databases containing compoundsalong with information on their molecular structure. Such a search seekscompounds having structures that match the determined active sitestructure and that interact with the groups defining the active site.Such a seach can be manual, but is preferably computer assisted.Compounds found from this search are potential ObR modulating compounds.

Alternatively, these methods can be used to identify improved modulatingcompounds from an already known modulating compound or ligand. Thecomposition of the known compound can be modified and the structuraleffects of modification can be determined using the experimental andcomputer modelling methods described above applied to the newcomposition. The altered structure is then compared to the active sitestructure of the compound to determine if an improved fit or interactionresults. In this manner, systematic variations in composition, such asby varying side groups, can be quickly evaluated to obtain modifiedmodulating compounds or ligands of improved specificity or activity.

Further experimental and computer modeling methods useful to identifymodulating compounds based upon identification of the active sites ofOb, ObR, and related transduction and transcription factors will beapparent to those of skill in the art.

Examples of molecular modelling systems are the CHARMm and QUANTAprograms (Polygen Corporation, Waltham, Mass.). CHARMm performs theenergy minimization and molecular dynamics functions. QUANTA performsthe construction, graphic modelling and analysis of molecular structure.QUANTA allows interactive construction, modification, visualization, andanalysis of the behavior of molecules with each other.

A number of articles review computer modelling of drugs interactive withspecific proteins, such as Rotivinen, et al., 1988, Acta PharmaceuticalFennica 97:159-166; Ripka, New Scientist 54-57 (Jun. 16, 1988); McKinalyand Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perryand Davies, OSAR: Quantitative Structure-Activity Relationships in DrugDesign pp. 189-193 (Alan R. Liss, Inc. 1989); Lewis and Dean, 1989,Proc. R. Soc. Lond. 236:125-140 and 141-162; and, with respect to amodel receptor for nucleic acid components, Askew et al., 1989, J. Am.Chem. Soc. 111:1082-1090. Other computer programs that screen andgraphically depict chemicals are available from companies such asBioDesign, Inc. (Pasadena, Calif.), Allelix, Inc. (Mississauga, Ontario,Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these areprimarily designed for application to drugs specific to particularproteins, they can be adapted to design of drugs specific to regions ofDNA or RNA, once that region is identified.

Although described above with reference to design and generation ofcompounds which could alter binding, one could also screen libraries ofknown compounds, including natural products or synthetic chemicals, andbiologically active materials, including proteins, for compounds whichare inhibitors or activators.

Compounds identified via assays such as those described herein may beuseful, for example, in elaborating the biological function of the obRgene product, and for ameliorating body weight disorders. Assays fortesting the effectiveness of compounds, identified by, for example,techniques such as those described in Section 5.5.1 through 5.5.3, arediscussed, below, in Section 5.5.4.

5.5.1. In Vitro Screening Assays for Compounds that Bind to ObR

In vitro systems may be designed to identify compounds capable ofinteracting with (e.g., binding to) ObR (including, but not limited to,the ECD or CD of ObR). Compounds identified can be used, for example, inmodulating the activity of wild type and/or mutant obR gene products; inelaborating the biological function of the ObR; in screens foridentifying compounds that disrupt normal ObR interactions; or can inthemselves disrupt such interactions.

The principle of the assays used to identify compounds that bind to theObR involves preparing a reaction mixture of the ObR and the testcompound under conditions and for a time sufficient to allow the twocomponents to interact and bind, thus forming a complex which can beremoved and/or detected in the reaction mixture. The ObR species usedcan vary depending upon the goal of the screening assay. For example,where agonists of the natural ligand are sought, the full length ObR, ora soluble truncated ObR, for example, in which the TM and/or CD isdeleted from the molecule, a peptide corresponding to the ECD or afusion protein containing the ObR ECD fused to a protein or polypeptidethat affords advantages in the assay system (e.g., labeling, isolationof the resulting complex, etc.) can be utilized. Where compounds thatinteract with the cytoplasmic domain are sought to be identified,peptides corresponding to the ObR CD and fusion proteins containing theObR CD can be used.

The screening assays can be conducted in a variety of ways. For example,one method to conduct such an assay would involve anchoring the ObRprotein, polypeptide, peptide or fusion protein or the test substanceonto a solid phase and detecting ObR/test compound complexes anchored onthe solid phase at the end of the reaction. In one embodiment of such amethod, the ObR reactant may be anchored onto a solid surface, and thetest compound, which is not anchored, may be labeled, either directly orindirectly.

In practice, microtiter plates may conveniently be utilized as the solidphase. The anchored component can be immobilized by non-covalent orcovalent attachments. Non-covalent attachment can be accomplished bysimply coating the solid surface with a solution of the protein anddrying. Alternatively, an immobilized antibody, preferably a monoclonalantibody, specific for the protein to be immobilized can be used toanchor the protein to the solid surface. The surfaces can be prepared inadvance and stored.

In order to conduct the assay, the nonimmobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynonimmobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously nonimmobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; forexample, using a labeled antibody specific for the previouslynonimmobilized component (the antibody, in turn, can be directly labeledor indirectly labeled with a labeled anti-Ig antibody).

Alternatively, a reaction can be conducted in a liquid phase, thereaction products separated from unreacted components, and complexesdetected; for example, using an immobilized antibody specific for ObRprotein, polypeptide, peptide or fusion protein or the test compound toanchor any complexes formed in solution, and a labeled antibody specificfor the other component of the possible complex to detect anchoredcomplexes.

Alternatively, cell-based assays can be used to identify compounds thatinteract with ObR. To this end, cell lines that express ObR, or celllines (e.g., COS cells, CHO cells, fibroblasts, etc.) that have beengenetically engineered to express ObR (e.g., by transfection ortransduction of ObR DNA) can be used. Interaction of the test compoundwith, for example, the ECD of obR expressed by the host cell can bedetermined by comparison or competition with native Ob.

5.5.2. Assays for Intracellular Proteins that Interact with the ObR

Any method suitable for detecting protein-protein interactions may beemployed for identifying transmembrane proteins or intracellularproteins that interact with ObR. Among the traditional methods which maybe employed are co-immunoprecipitation, crosslinking and co-purificationthrough gradients or chromatographic columns of cell lysates or proteinsobtained from cell lysates and the ObR to identify proteins in thelysate that interact with the ObR. For these assays, the ObR componentused can be a full length ObR, a soluble derivative lacking themembrane-anchoring region (e.g., a truncated ObR in which the TM isdeleted resulting in a truncated molecule containing the ECD fused tothe CD), a peptide corresponding to the CD or a fusion proteincontaining the CD of ObR. Once isolated, such an intracellular proteincan be identified and can, in turn, be used, in conjunction withstandard techniques, to identify proteins with which it interacts. Forexample, at least a portion of the amino acid sequence of anintracellular protein which interacts with the ObR can be ascertainedusing techniques well known to those of skill in the art, such as viathe Edman degradation technique. (See, e.g., Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., pp.34-49). The amino acid sequence obtained may be used as a guide for thegeneration of oligonucleotide mixtures that can be used to screen forgene sequences encoding such intracellular proteins. Screening may beaccomplished, for example, by standard hybridization or PCR techniques.Techniques for the generation of oligonucleotide mixtures and thescreening are well-known. (See, e.g., Ausubel, supra, and PCR Protocols:A Guide to Methods and Applications, 1990, Innis, M. et al., eds.Academic Press, Inc., New York).

Additionally, methods may be employed that result in the simultaneousidentification of genes which encode the transmembrane or intracellularproteins interacting with ObR. These methods include, for example,probing expression libraries, in a manner similar to the well knowntechnique of antibody probing of λgt11 libraries, using labeled ObRprotein, or an ObR polypeptide, peptide, or fusion protein, e.g., an ObRpolypeptide or ObR domain fused to a marker (e.g., an enzyme,fluorophore, luminescent protein, or dye), or an Ig-Fc domain.

One method that detects protein interactions in vivo, the two-hybridsystem, is described in detail for illustration only and not by way oflimitation. One version of this system has been described (Chien et al.,1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582) and is commerciallyavailable from Clontech (Palo Alto, Calif.).

Briefly, utilizing such a system, plasmids are constructed that encodetwo hybrid proteins: one plasmid consists of nucleotides encoding theDNA-binding domain of a transcription activator protein fused to an obRnucleotide sequence encoding ObR, an ObR polypeptide, peptide, or fusionprotein, and the other plasmid consists of nucleotides encoding thetranscription activator protein's activation domain fused to a cDNAencoding an unknown protein which has been recombined into this plasmidas part of a cDNA library. The DNA-binding domain fusion plasmid and thecDNA library are transformed into a strain of the yeast Saccharomycescerevisiae that contains a reporter gene (e.g., HBS or lacZ) whoseregulatory region contains the transcription activator's binding site.Either hybrid protein alone cannot activate transcription of thereporter gene: the DNA-binding domain hybrid cannot because it does notprovide activation function and the activation domain hybrid cannotbecause it cannot localize to the activator's binding sites. Interactionof the two hybrid proteins reconstitutes the functional activatorprotein and results in expression of the reporter gene, which isdetected by an assay for the reporter gene product.

The two-hybrid system or related methodology can be used to screenactivation domain libraries for proteins that interact with the “bait”gene product. By way of example, and not by way of limitation, ObR maybe used as the bait gene product. Total genomic or cDNA sequences arefused to the DNA encoding an activation domain. This library and aplasmid encoding a hybrid of a bait obR gene product fused to theDNA-binding domain are cotransformed into a yeast reporter strain, andthe resulting transformants are screened for those that express thereporter gene. For example, and not by way of limitation, a bait obRgene sequence, such as the open reading frame of obR (or a domain ofobR), as depicted in FIGS. 1A-1D, FIGS. 3A-3F, or FIGS. 6A-6F can becloned into a vector such that it is translationally fused to the DNAencoding the DNA-binding domain of the GAL4 protein. These colonies arepurified and the library plasmids responsible for reporter geneexpression are isolated. DNA sequencing is then used to identify theproteins encoded by the library plasmids.

A cDNA library of the cell line from which proteins that interact withbait obR gene product are to be detected can be made using methodsroutinely practiced in the art. According to the particular systemdescribed herein, for example, the cDNA fragments can be inserted into avector such that they are translationally fused to the transcriptionalactivation domain of GAL4. This library can be co-transformed along withthe bait obR gene-GAL4 fusion plasmid into a yeast strain which containsa lacZ gene driven by a promoter which contains GAL4 activationsequence. A cDNA encoded protein, fused to GAL4 transcriptionalactivation domain, that interacts with bait obR gene product willreconstitute an active GAL4 protein and thereby drive expression of theHIS3 gene. Colonies which express HIS3 can be detected by their growthon petri dishes containing semi-solid agar based media lackinghistidine. The cDNA can then be purified from these strains, and used toproduce and isolate the bait obR gene-interacting protein usingtechniques routinely practiced in the art.

5.5.3. Assays for Compounds that Interfere with ObR/Intracellular orObR/Transmembrane Macromolecule Interaction

The macromolecules that interact with the ObR are referred to, forpurposes of this discussion, as “binding partners.” These bindingpartners are likely to be involved in the ObR signal transductionpathway, and therefore, in the role of ObR in body weight regulation.Therefore, it is desirable to identify compounds that interfere with ordisrupt the interaction of such binding partners with Ob. Thesecompounds can be used, for example, to regulate the activity of the ObR,and thereby control body weight disorders associated with ObR activity.

The basic principle of the assay systems used to identify compounds thatinterfere with the interaction between the ObR and its binding partneror partners involves preparing a reaction mixture containing ObRprotein, polypeptide, peptide, or fusion protein as described inSections 5.5.1 and 5.5.2 above, and the binding partner under conditionsand for a time sufficient to allow the two to interact and bind, thusforming a complex. In order to test a compound for inhibitory activity,the reaction mixture is prepared in the presence and absence of the testcompound. The test compound may be initially included in the reactionmixture, or may be added at a time subsequent to the addition of the ObRmoiety and its binding partner. Control reaction mixtures are incubatedwithout the test compound or with a placebo. The formation of anycomplexes between the ObR moiety and the binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the ObR and the interactivebinding partner. Additionally, complex formation within reactionmixtures containing the test compound and normal ObR protein may also becompared to complex formation within reaction mixtures containing thetest compound and a mutant ObR. This comparison may be important inthose cases wherein it is desirable to identify compounds that disruptinteractions of mutant but not normal ObRs.

The assay for compounds that interfere with the interaction of the ObRand binding partners can be conducted in a heterogeneous or homogeneousformat. Heterogeneous assays involve anchoring either the ObR moietyproduct or the binding partner onto a solid phase and detectingcomplexes anchored on the solid phase at the end of the reaction. Inhomogeneous assays, the entire reaction is carried out in a liquidphase. In either approach, the order of addition of reactants can bevaried to obtain different information about the compounds being tested.For example, test compounds that interfere with the interaction bycompetition can be identified by conducting the reaction in the presenceof the test substance; i.e., by adding the test substance to thereaction mixture prior to or simultaneously with the ObR moiety andinteractive binding partner. Alternatively, test compounds that disruptpreformed complexes, for example, compounds with higher bindingconstants that displace one of the components from the complex, can betested by adding the test compound to the reaction mixture aftercomplexes have been formed. The various formats are described brieflybelow.

In a heterogeneous assay system, either the ObR moiety or theinteractive binding partner, is anchored onto a solid surface, while thenon-anchored species is labeled, either directly or indirectly. Inpractice, microtiter plates are conveniently utilized. The anchoredspecies can be immobilized by non-covalent or covalent attachments.Non-covalent attachment can be accomplished simply by coating the solidsurface with a solution of the obR gene product or binding partner anddrying. Alternatively, an immobilized antibody specific for the speciesto be anchored can be used to anchor the species to the solid surface.The surfaces can be prepared in advance and stored.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. The detection of complexes anchored on the solid surface can beaccomplished in a number of ways. Where the non-immobilized species ispre-labeled, the detection of label immobilized on the surface indicatesthat complexes were formed. Where the non-immobilized species is notpre-labeled, an indirect label can be used to detect complexes anchoredon the surface; for example, using a labeled antibody specific for theinitially non-immobilized species (the antibody, in turn, can bedirectly labeled or indirectly labeled with a labeled anti-Ig antibody).Depending upon the order of addition of reaction components, testcompounds which inhibit complex formation or which disrupt preformedcomplexes can be detected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; forexample, using an immobilized antibody specific for one of the bindingcomponents to anchor any complexes formed in solution, and a labeledantibody specific for the other partner to detect anchored complexes.Again, depending upon the order of addition of reactants to the liquidphase, test compounds that inhibit complex or which disrupt preformedcomplexes can be identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. In this approach, a preformed complex of the ObR moiety and theinteractive binding partner is prepared in which either the ObR or itsbinding partner is labeled, but the signal generated by the label isquenched due to formation of the complex (see, e.g., U.S. Pat. No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).The addition of a test substance that competes with and displaces one ofthe species from the preformed complex will result in the generation ofa signal above background. In this way, test substances which disruptObR/intracellular binding partner interaction can be identified.

In a particular embodiment, an ObR fusion can be prepared forimmobilization. For example, the ObR or a peptide fragment, for example,corresponding to the CD, can be fused to a glutathione-S-transferase(GST) gene using a fusion vector, such as pGEX-5X-1, in such a mannerthat its binding activity is maintained in the resulting fusion protein.The interactive binding partner can be purified and used to raise amonoclonal antibody, using methods routinely practiced in the art anddescribed above, in Section 5.3. This antibody can be labeled with theradioactive isotope ¹²⁵I, for example, by methods routinely practiced inthe art. In a heterogeneous assay, for example, the GST-ObR fusionprotein can be anchored to glutathione-agarose beads. The interactivebinding partner can then be added in the presence or absence of the testcompound in a manner that allows interaction and binding to occur. Atthe end of the reaction period, unbound material can be washed away, andthe labeled monoclonal antibody can be added to the system and allowedto bind to the complexed components. The interaction between the obRgene product and the interactive binding partner can be detected bymeasuring the amount of radioactivity that remains associated with theglutathione-agarose beads. A successful inhibition of the interaction bythe test compound will result in a decrease in measured radioactivity.

Alternatively, the GST-ObR fusion protein and the interactive bindingpartner can be mixed together in liquid in the absence of the solidglutathione-agarose beads. The test compound can be added either duringor after the species are allowed to interact. This mixture can then beadded to the glutathione-agarose beads and unbound material is washedaway. Again the extent of inhibition of the ObR/binding partnerinteraction can be detected by adding the labeled antibody and measuringthe radioactivity associated with the beads.

In another embodiment of the invention, these same techniques can beemployed using peptide fragments that correspond to the binding domainsof the ObR and/or the interactive or binding partner (in cases where thebinding partner is a protein), in place of one or both of the fulllength proteins. Any number of methods routinely practiced in the artcan be used to identify and isolate the binding sites. These methodsinclude, but are not limited to, mutagenesis of the gene encoding one ofthe proteins and screening for disruption of binding in aco-immunoprecipitation assay. Compensating mutations in the geneencoding the second species in the complex can then be selected.Sequence analysis of the genes encoding the respective proteins willreveal the mutations that correspond to the region of the proteininvolved in interactive binding. Alternatively, one protein can beanchored to a solid surface using methods described above, and allowedto interact with and bind to its labeled binding partner, which has beentreated with a proteolytic enzyme, such as trypsin. After washing, ashort, labeled peptide comprising the binding domain may remainassociated with the solid material, which can be isolated and identifiedby amino acid sequencing. Also, once the gene coding for theintracellular binding partner is obtained, short gene segments can beengineered to express peptide fragments of the protein, which can thenbe tested for binding activity and purified or synthesized.

For example, and not by way of limitation, an obR gene product can beanchored to a solid material, as described above, by making a GST-ObRfusion protein and allowing it to bind to glutathione agarose beads. Theinteractive binding partner can be labeled with a radioactive isotope,such as ³⁵S, and cleaved with a proteolytic enzyme such as trypsin.Cleavage products can then be added to the anchored GST-obR fusionprotein and allowed to bind. After washing away unbound peptides,labeled bound material, representing the intracellular binding partnerbinding domain, can be eluted, purified, and analyzed for amino acidsequence by well-known methods. Peptides so identified can be producedsynthetically or fused to appropriate facilitative proteins usingrecombinant DNA technology.

5.5.4. Assays for Identification of Compounds that Ameliorate BodyWeight Disorders

Compounds, including but not limited to binding compounds identified viaassay techniques such as those described above, in Sections 5.5.1through 5.5.3, can be tested for the ability to ameliorate body weightdisorder symptoms, including obesity. The assays described above canidentify compounds that affect ObR activity (e.g., compounds that bindto the ObR, inhibit binding of the natural ligand, and either activatesignal transduction (agonists) or block activation (antagonists), andcompounds that bind to the natural ligand of the ObR and neutralizeligand activity); or compounds that affect obR gene activity (byaffecting obR gene expression, including molecules, e.g., proteins orsmall organic molecules, that affect or interfere with splicing eventsso that expression of the full length or the truncated form of the ObRcan be modulated). However, it should be noted that the assays describedcan also identify compounds that modulate ObR signal transduction (e.g.,compounds which affect downstream signalling events, such as inhibitorsor enhancers of tyrosine kinase or phosphatase activities whichparticipate in transducing the signal activated by Ob binding to theObR). The identification and use of such compounds which affect anotherstep in the ObR signal transduction pathway in which the obR gene and/orobR gene product is involved and, by affecting this same pathway canmodulate the effect of ObR on the development of body weight disordersare within the scope of the invention. Such compounds can be used aspart of a therapeutic method for the treatment of body weight disorders.

The invention encompasses cell-based and animal model-based assays forthe identification of compounds exhibiting such an ability to amelioratebody weight disorder symptoms. Such cell-based assay systems can also beused as the “gold standard” to assay for purity and potency of thenatural ligand, Ob, including recombinantly or synthetically produced Oband Ob mutants.

Cell-based systems can be used to identify compounds that can act toameliorate body weight disorder symptoms. Such cell systems can include,for example, recombinant or non-recombinant cells, such as cell lines,which express the obR gene. For example choroid plexus cells,hypothalamus cells, or cell lines derived from choroid plexus orhypothalamus can be used. In addition, expression host cells (e.g., COScells, CHO cells, fibroblasts) genetically engineered to express afunctional ObR and to respond to activation by the natural Ob ligand,e.g., as measured by a chemical or phenotypic change, induction ofanother host cell gene, change in ion flux (e.g., Ca⁺⁺), tyrosinephosphorylation of host cell proteins, etc., can be used as an end pointin the assay.

In utilizing such cell systems, cells may be exposed to a compoundsuspected of exhibiting an ability to ameliorate body weight disordersymptoms, at a sufficient concentration and for a time sufficient toelicit such an amelioration of body weight disorder symptoms in theexposed cells. After exposure, the cells can be assayed to measurealterations in the expression of the obR gene, for example, by assayingcell lysates for obR mRNA transcripts (e.g., by Northern analysis) orfor obR protein expressed in the cell; compounds that regulate ormodulate expression of the obR gene are good candidates as therapeutics.Alternatively, the cells are examined to determine whether one or morebody weight disorder-like cellular phenotypes has been altered toresemble a more normal or more wild type, non-body weight disorderphenotype, or a phenotype more likely to produce a lower incidence orseverity of disorder symptoms. Still further, the expression and/oractivity of components of the signal transduction pathway of which ObRis a part, or the activity of the ObR signal transduction pathway itselfcan be assayed.

For example, after exposure, the cell lysates can be assayed for thepresence of tyrosine phosphorylation of host cell proteins, as comparedto lysates derived from unexposed control cells. The ability of a testcompound to inhibit tyrosine phosphorylation of host cell proteins inthese assay systems indicates that the test compound inhibits signaltransduction initiated by ObR activation. The cell lysates can bereadily assayed using a Western blot format; i.e., the host cellproteins are resolved by gel electrophoresis, transferred to a support,and probed using a anti-phosphotyrosine detection antibody (e.g., ananti-phosphotyrosine antibody labeled with a signal generating compound,such as radiolabel, fluorophore, enzyme, etc.) (See, e.g., Glenney etal., 1988, J. Immunol. Methods 109:277-285; Frackelton et al., 1983,Mol. Cell. Biol. 3:1343-1352). Alternatively, an ELISA format could beused in which a particular host cell protein involved in the ObR signaltransduction pathway is immobilized using an anchoring antibody specificfor the target host cell protein, and the presence or absence ofphosphotyrosine on the immobilized host cell protein is detected using alabeled anti-phosphotyrosine antibody. (See, King et al., 1993, LifeSciences 53:1465-1472). In yet another approach, ion flux, such ascalcium ion flux, can be measured as an end point for ObR stimulatedsignal transduction.

Alternatively, activation of STAT proteins, and stimulation oftranscription mediated through IL-6 responsive gene elements can bemeasured to test the ability of a compound to regulate ObR mediatedsignal transduction. For example, a recombinant expression vector can beengineered to contain the IL-6 responsive element sequences clonedadjacent to a reporter gene and regulation of ObR activity may bemeasured by assaying for reporter gene activity. Reporter genes that maybe used include, but are not limited to those encoding chloramphenicolacetyl transferase (CAT), firefly luciferase, or human growth hormone.

In addition, animal-based body weight disorder systems, which mayinclude, for example, ob, db and ob/db mice, can be used to identifycompounds capable of ameliorating body weight disorder-like symptoms.Such animal models may be used as test substrates for the identificationof drugs, pharmaceuticals, therapies and interventions which can beeffective in treating such disorders. For example, animal models can beexposed to a compound, suspected of exhibiting an ability to amelioratebody weight disorder symptoms, at a sufficient concentration and for atime sufficient to elicit such an amelioration of body weight disordersymptoms in the exposed animals. The response of the animals to theexposure may be monitored by assessing the reversal of disordersassociated with body weight disorders such as obesity. With regard tointervention, any treatments which reverse any aspect of body weightdisorder-like symptoms would be considered as candidates for human bodyweight disorder therapeutic intervention. Dosages of test agents may bedetermined by deriving dose-response curves, as discussed in Section5.7.1, below.

5.6. The Treatment of Body Weight, Including Body Weight Disorders

The invention encompasses methods and compositions for modifying bodyweight and treating body weight disorders, including but not limited toobesity, cachexia and anorexia. Because a loss of normal obR geneproduct function results in the development of an obese phenotype, anincrease in obR gene product activity, or activation of the ObR pathway(e.g., downstream activation) would facilitate progress towards a normalbody weight state in obese individuals exhibiting a deficient level ofobR gene expression and/or obR activity.

Alternatively, symptoms of certain body weight disorders such as, forexample, cachexia, which involve a lower than normal body weightphenotype, may be ameliorated by decreasing the level of obR geneexpression, and/or obR gene activity, and/or downregulating activity ofthe ObR pathway (e.g., by targeting downstream signalling events).Different approaches are discussed below.

-   5.6.1. Inhibition of ObR Expression or ObR Activity to Promote    Weight Gain

Any method that neutralizes Ob or inhibits expression of the obR gene(either transcription or translation) can be used to effectuate weightgain. Such approaches can be used to treat body weight disorders such asanorexia or cachexia. Such methods can also be useful for agriculturalapplications; i.e., to increase the weight of livestock animals.

For example, the administration of soluble peptides, proteins, fusionproteins, or antibodies (including anti-idiotypic antibodies) that bindto and “neutralize” circulating Ob, the natural ligand for the ObR, canbe used to effectuate weight gain. To this end, peptides correspondingto the ECD of ObR, soluble deletion mutants of ObR (e.g., ΔTMObRmutants), or either of these ObR domains or mutants fused to anotherpolypeptide (e.g., an IgFc polypeptide) can be utilized. Alternatively,anti-idiotypic antibodies or Fab fragments of antiidiotypic antibodiesthat mimic the ObR ECD and neutralize Ob can be used (see Section 5.3,supra). Such ObR peptides, proteins, fusion proteins, anti-idiotypicantibodies or Fabs are administered to a subject in amounts sufficientto neutralize Ob and to effectuate weight gain.

ObR peptides corresponding to the ECD having the amino acid sequenceshown in FIG. 1A-1D or 6A-6F, from about amino acid residue 23 to aboutamino acid residue 837, or having the amino acid sequence shown in FIGS.3A-3F, from about amino acid residue 21 to about amino acid residue 839,can be used. ObR ΔTM mutants in which all or part of the 23 amino acidhydrophobic anchor sequence (e.g., about amino acid residue 838 to aminoacid residue 860 in FIG. 1A-1D or 6A-6F, or about amino acid residue 840to about amino acid residue 862 in FIGS. 3A-3F) could also be used.Fusion of the ObR, the ObR ECD or the ΔTMObR to an IgFc polypeptideshould not only increase the stability of the preparation, but willincrease the half-life and activity of the ObR-Ig fusion protein invivo. The Fc region of the Ig portion of the fusion protein can befurther modified to reduce immunoglobulin effector function. See Section10, infra.

In a specific embodiment described herein, the extracellular domains ofthe mouse or human ObR were fused to the IgG constant region. Asindicated in FIG. 10, purified ObR-IgG was able to potently inhibit, orneutralize, the binding of the AP-OB fusion protein to cell surface ObR(See Section 10.4).

In an alternative embodiment for neutralizing circulating Ob, cells thatare genetically engineered to express such soluble or secreted forms ofObR can be administered to a patient, whereupon they will serve as“bioreactors” in vivo to provide a continuous supply of the Obneutralizing protein. Such cells may be obtained from the patient or anMHC compatible donor and can include, but are not limited tofibroblasts, blood cells (e.g., lymphocytes), adipocytes, muscle cells,endothelial cells, etc. The cells are genetically engineered in vitrousing recombinant DNA techniques to introduce the coding sequence forthe ObR ECD, ΔTMObR, or for ObR-Ig fusion protein (e.g., ObR-, ECD- orΔTMObR-IgFc fusion proteins) into the cells, for example, bytransduction (using viral vectors, and preferably vectors that integratethe transgene into the cell genome) or transfection procedures,including but not limited to the use of plasmids, cosmids, YACs,electroporation, liposomes, etc. The obR coding sequence can be placedunder the control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression and secretion of the ObR peptideor fusion protein. The engineered cells that express and secrete thedesired ObR product can be introduced into the patient systemically, forexample, in the circulation, intraperitoneally, at the choroid plexus,or hypothalamus. Alternatively, the cells can be incorporated into amatrix and implanted in the body. For example, genetically engineeredfibroblasts can be implanted as part of a skin graft; geneticallyengineered endothelial cells can be implanted as part of a vasculargraft. (See, for example, Anderson et al. U.S. Pat. No. 5,399,349; andMulligan & Wilson, U.S. Pat. No. 5,460,959 each of which is incorporatedby reference herein in its entirety).

When the cells to be administered are non-autologous cells, they can beadministered using well known techniques that prevent the development ofa host immune response against the introduced cells. For example, thecells can be introduced in an encapsulated form which, while allowingfor an exchange of components with the immediate extracellularenvironment, does not allow the introduced cells to be recognized by thehost immune system.

In an alternate embodiment, weight gain therapy can be designed toreduce the level of endogenous obR gene expression, for example, usingantisense or ribozyme approaches to inhibit or prevent translation ofobR mRNA transcripts; triple helix approaches to inhibit transcriptionof the obR gene; or targeted homologous recombination to inactivate or“knock out” the obR gene or its endogenous promoter. Because the obRgene is expressed in the brain, including the choroid plexus andhypothalamus, delivery techniques should be preferably designed to crossthe blood-brain barrier (see PCT WO89/10134, which is incorporated byreference herein in its entirety). Alternatively, the antisense,ribozyme or DNA constructs described herein could be administereddirectly to the site containing the target cells; for example, thechoroid plexus and/or hypothalamus.

Antisense approaches involve the design of oligonucleotides (either DNAor RNA) that are complementary to ObR mRNA. The antisenseoligonucleotides will bind to the complementary obR mRNA transcripts andprevent translation. Absolute complementarity, although preferred, isnot required. A sequence “complementary” to a portion of an RNA, asreferred to herein, means a sequence having sufficient complementarityto be able to hybridize with the RNA, forming a stable duplex; in thecase of double-stranded antisense nucleic acids, a single strand of theduplex DNA may thus be tested, or triplex formation may be assayed. Theability to hybridize will depend on both the degree of complementarityand the length of the antisense nucleic acid. Generally, the longer thehybridizing nucleic acid, the more base mismatches with an RNA it maycontain and still form a stable duplex (or triplex, as the case may be).One skilled in the art can ascertain a tolerable degree of mismatch byuse of standard procedures to determine the melting point of thehybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,for example, the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have recently shown to be effective at inhibitingtranslation of mRNAs as well, See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of the obR shown in FIGS. 1A-1D(murine short form), FIGS. 6A-6F (murine long form) or FIGS. 3A-3F(human long form) could be used in an anti sense approach to inhibittranslation of endogenous obR mRNA. Oligonucleotides complementary tothe 5′ untranslated region of the mRNA should include the complement ofthe AUG start codon. Antisense oligonucleotides complementary to mRNAcoding regions are less efficient inhibitors of translation but could beused in accordance with the invention. Whether designed to hybridize tothe 5′-, 3′- or coding region of ObR mRNA, antisense nucleic acidsshould be at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects, the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides, or at least 50nucleotides in length.

Regardless of the choice of target sequence, it is preferred that invitro studies are first performed to quantitate the ability of theantisense oligonucleotide to inhibit gene expression. It is preferredthat these studies utilize controls that distinguish between antisensegene inhibition and nonspecific biological effects of oligonucleotides.It is also preferred that these studies compare levels of the target RNAor protein with that of an internal control RNA or protein.Additionally, it is envisioned that results obtained using the antisenseoligonucleotide are compared with those obtained using a controloligonucleotide. It is preferred that the control oligonucleotide is ofapproximately the same length as the test oligonucleotide and that thenucleotide sequence of the oligonucleotide differs from the antisensesequence no more than is necessary to prevent specific hybridization tothe target sequence.

The oligonucleotides can be DNA or RNA or chimeric mixtures orderivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide can includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci. USA86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. USA84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide can be conjugated to another molecule, for example, apeptide, hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including but not limited to5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide can also comprise at least one modifiedsugar moiety selected from the group including but not limited toarabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group consistingof a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, aphosphoramidate, a phosphordiamidate, a methylphosphonate, an alkylphosphotriester, and a formacetal or analog thereof.

In yet another embodiment, the antisense oligonucleotide is anα-anomeric oligonucleotide. An α-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual β-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Oligonucleotides of the invention can be synthesized by standard methodsknown in the art, for example, by use of an automated DNA synthesizer(such as are commercially available from Biosearch, Applied Biosystems,etc.). As examples, phosphorothioate oligonucleotides can be synthesizedby the method of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.USA 85:7448-7451), etc.

While antisense nucleotides complementary to the obR coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred. For example, antisenseoligonucleotides having the following sequences can be utilized inaccordance with the invention:

-   a) 5′-CATCTTACTTCAGAGAA-3′ (SEQ ID NO:7), which is complementary to    nucleotides −14 to +3 in FIGS. 3A-3F;-   b) 5′-CATCTTACTTCAGAGAAGTACAC-3′ (SEQ ID NO:8), which is    complementary to nucleotides −20 to +3 in FIGS. 3A-3F;-   c) 5′-CATCTTACTTCAGAGAAGTACACCCATAA-3′ (SEQ ID NO:9), which is    complementary to nucleotides −26 to +3 in FIGS. 3A-3F;-   d) 5′-CATCTTACTTCAGAGAAGTACACCCATAATCCTCT-3′ (SEQ ID NO:10), which    is complementary to nucleotides −32 to +3 in FIGS. 3A-3F;-   e) 5′-AATCATCTTACTTCAGAGAAGTACACCCATAATCC-3′ (SEQ ID NO: 11), which    is complementary to nucleotides −29 to +6 in FIGS. 3A-3F;-   f) 5′-CTTACTTCAGAGAAGTACACCCATAATCC-3′ (SEQ ID NO:12), which is    complementary to nucleotides −29 to −1 in FIGS. 3A-3F;-   g) 5′-TCACGAGAAGTACACCCATAATCC-3′ (SEQ ID NO: 13), which is    complementary to nucleotides −29 to −7 in FIGS. 3A-3F;-   h) 5-AAGTACACCCATAATCC-3′ (SEQ ID NO:14), which is complementary to    nucleotides −29 to −13 in FIGS. 3A-3F.

The antisense molecules should be delivered to cells which express theObR in vivo, e.g., the choroid plexus and/or hypothalamus. A number ofmethods have been developed for delivering antisense DNA or RNA tocells; for example, antisense molecules can be injected directly intothe tissue site, or modified antisense molecules, designed to target thedesired cells (e.g., antisense linked to peptides or antibodies thatspecifically bind receptors or antigens expressed on the target cellsurface) can be administered systemically.

However, it is often difficult to achieve intracellular concentrationsof the antisense sufficient to suppress translation of endogenous mRNAs.Therefore, a preferred approach utilizes a recombinant DNA construct inwhich the antisense oligonucleotide is placed under the control of astrong pol III or pol II promoter. The use of such a construct totransfect target cells in the patient will result in the transcriptionof sufficient amounts of single stranded RNAs that will formcomplementary base pairs with the endogenous obR transcripts and therebyprevent translation of the obR mRNA. For example, a vector can beintroduced in vivo such that it is taken up by a cell and directs thetranscription of an antisense RNA. Such a vector can remain episomal orbecome chromosomally integrated, as long as it can be transcribed toproduce the desired antisense RNA. Such vectors can be constructed byrecombinant DNA technology methods standard in the art. Vectors can beplasmid vectors, viral vectors, or other vectors known in the art to beuseful for replication and expression of nucleic acids in mammaliancells. Expression of the sequence encoding the antisense RNA can be byany promoter known in the art to act in mammalian, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude but are not limited to: the SV40 early promoter region (Bernoistand Chambon, 1981, Nature 290:304-310), the promoter contained in the 3′long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell22:787-797), the herpes thymidine kinase promoter (Wagner et al., 1981,Proc. Natl. Acad. Sci. USA 78:1441-1445), the regulatory sequences ofthe metallothionein gene (Brinster et al., 1982, Nature 296:39-42), etc.Any type of plasmid, cosmid, YAC, or viral vector can be used to preparethe recombinant DNA construct which can be introduced directly into thetissue site; for example, the choroid plexus or hypothalamus.Alternatively, viral vectors can be used which selectively infect thedesired tissue; (e.g., for brain, herpesvirus vectors may be used), inwhich case administration may be accomplished by another route (e.g.,systemically).

Ribozyme molecules designed to catalytically cleave obR mRNA transcriptscan also be used to prevent translation of obR mRNA and expression ofObR. (See, e.g., PCT International Publication WO90/11364, publishedOct. 4, 1990; Sarver et al., 1990, Science 247:1222-1225). Whileribozymes that cleave mRNA at site specific recognition sequences can beused to destroy obR mRNAs, the use of hammerhead ribozymes is preferred.Hammerhead ribozymes cleave mRNAs at locations dictated by flankingregions that form complementary base pairs with the target mRNA. Thesole requirement is that the target mRNA have the following sequence oftwo bases: 5′-UG-3′. The construction and production of hammerheadribozymes is well known in the art and is described more fully inHaseloff and Gerlach, 1988, Nature 334:585-591. There are hundreds ofpotential hammerhead ribozyme cleavage sites within the nucleotidesequence of human obR cDNA FIGS. 3A-3F). Preferably the ribozyme isengineered so that the cleavage recognition site is located near the 5′end of the obR mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

For example, hammerhead ribozymes having the following sequences can beutilized in accordance with the invention:

-   a) 5′-ACAGAAUUUUUGACAAAUCAAAGCAGANNNNUCUGAGNAGUCCUUACUUCAGAGAA-3′    (SEQ ID NO:15), which will cleave human obR mRNA between nucleotides    −1 and 1 in FIGS. 3A-3F;-   b) 5-GGCCCGGGCAGCCUGCCCAAAGCCGGNNNNCCGGAGNAGUCGCCAGACCGGCUCGUG-3′    (SEQ ID NO:16), which will cleave between nucleotides −175 and −176    in FIGS. 3A-3F;-   c) 5′-UGGCAUGCAAGACAAAGCAGGNNNNCCUGAGNAGUCCUUAAAUCUCCAAGGAGUAA-3′    (SEQ ID NO:17), which will cleave between nucleotides 102 and 103 in    FIGS. 3A-3F;-   d) 5′-UAUAUGACAAAGCUGUNNNNACAGAGNAGUCCUUGUGUGGUAAAGACACG-3′ (SEQ ID    NO:18), which will cleave between nucleotides 994 and 995 in FIGS.    3A-3F;-   e)    5′-AGCACCAAUUGAAUUGAUGGCCAAAGCGGGNNNNCCCGAGNAGUCAACCGUAACAGUAUGU-3′    (SEQ ID NO:19), which will cleave between nucleotides 2142 and 2143    in FIGS. 3A-3F;-   f)    5′-UGAAAUUGUUUCAGGCUCCAAAGCCGGNNNNCCGGAGNAGUCAAGAAGAGGACCACAUGUCACUGAUGC-3′    (SEQ ID NO:20), which will cleave between nucleotides 2736 and 2737    in FIGS. 3A-3F;-   g)    5′-GGUUUCUUCAGUGAAAUUACACAAAGCAGCNNNNGCUGAGNAGUCAGUUAGGUCACACAUC-3′    (SEQ ID NO:21), which will cleave between nucleotides 3492 and 3493    in FIGS. 3A-3F;-   h) 5′-ACCCAUUAUAACACAAAGCUGANNNNUCAGAGNAGUCAUCUGAAGGUUUCUUC-3′ (SEQ    ID NO:22), which will cleave between nucleotides 3521 and 3522 in    FIGS. 3A-3F.

The ribozymes of the present invention also include RNAendoribonucleases (hereinafter “Cech-type ribozymes”) such as the onewhich occurs naturally in Tetrahymena Thermophila (known as the IVS, orL-19 IVS RNA) and which has been extensively described by Thomas Cechand collaborators (Zaug, et al., 1984, Science 224:574-578; Zaug andCech, 1986, Science 231:470-475; Zaug, et al., 1986, Nature 324:429-433;published International patent application No. WO 88/04300 by UniversityPatents Inc.; Been and Cech, 1986, Cell 47:207-216). The Cech-typeribozymes have an eight basepair active site that hybridizes to a targetRNA sequence, whereafter cleavage of the target RNA takes place. Theinvention encompasses those Cech-type ribozymes that target eightbasepair active site sequences that are present in obR.

As in the antisense approach, the ribozymes can be composed of modifiedoligonucleotides (e.g. for improved stability, targeting, etc.) andshould be delivered to cells which express the ObR in vivo, for example,hypothalamus and/or the choroid plexus. A preferred method of deliveryinvolves using a DNA construct “encoding” the ribozyme under the controlof a strong constitutive pol III or pol II promoter, so that transfectedcells will produce sufficient quantities of the ribozyme to destroyendogenous obR messages and inhibit translation. Because ribozymes,unlike antisense molecules, are catalytic, a lower intracellularconcentration is required for efficiency.

Endogenous obR gene expression can also be reduced by inactivating or“knocking out” the obR gene or its promoter using targeted homologousrecombination. (E.g., see Smithies et al., 1985, Nature 317:230-234;Thomas & Capecchi, 1987, Cell 51:503-512; Thompson et al., 1989, Cell5:313-321; each of which is incorporated by reference herein in itsentirety). For example, a mutant, non-functional ObR (or a completelyunrelated DNA sequence) flanked by DNA homologous to the endogenous obRgene (either the coding regions or regulatory regions of the obR gene)can be used, with or without a selectable marker and/or a negativeselectable marker, to transfect cells that express ObR in vivo.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the obR gene. Such approaches areparticularly suited in the agricultural field where modifications to ES(embryonic stem) cells can be used to generate animal offspring with aninactive ObR (e.g., see Thomas & Capecchi, 1987, supra and Thompson etal., 1989, supra). However, this approach can be adapted for use inhumans provided the recombinant DNA constructs are directly administeredor targeted to the required site in vivo using appropriate viralvectors, for example, herpes virus vectors for delivery to brain tissue;for example, the hypothalamus and/or choroid plexus.

Alternatively, endogenous obR gene expression can be reduced bytargeting deoxyribonucleotide sequences complementary to the regulatoryregion of the obR gene (i.e., the obR promoter and/or enhancers) to formtriple helical structures that prevent transcription of the obR gene intarget cells in the body. (See generally, Helene, C. 1991, AnticancerDrug Des. 6 (6):569-84; Helene, C. et al., 1992, Ann. N.Y. Acad. Sci.660:27-36; and Maher, L. J., 1992, Bioassays 14 (12):807-15).

In yet another embodiment of the invention, the activity of ObR can bereduced using a “dominant negative” approach to effectuate weight gain.To this end, constructs that encode defective ObRs can be used in genetherapy approaches to diminish the activity of the ObR in appropriatetarget cells. For example, nucleotide sequences that direct host cellexpression of ObRs in which the CD (e.g. FIGS. 1A-1D, amino acidresidues 861-894; FIGS. 6A-6F, amino acid residues 861-1162; or FIGS.3A-3F, amino acid residues 863-1165), or a portion of the CD (e.g., thebox 1 Jak interaction sequence; FIGS. 1A-1D and 6A-6F, amino acidresidues 861-884; or FIGS. 3A-3F, amino acid residues 863-886) isdeleted or mutated can be introduced into cells in the choroid plexus orhypothalamus (either by in vivo or ex vivo gene therapy methodsdescribed above). Alternatively, targeted homologous recombination canbe utilized to introduce such deletions or mutations into the subject'sendogenous obR gene in the hypothalamus or choroid plexus. Theengineered cells will express non-functional receptors (i.e., ananchored receptor that is capable of binding its natural ligand, butincapable of signal transduction). Such engineered cells present in thechoroid plexus or hypothalamus should demonstrate a diminished responseto the endogenous Ob ligand, resulting in weight gain.

5.6.2. Restoration or Increase in ObR Expression or Activity to PromoteWeight Loss

With respect to an increase in the level of normal obR gene expressionand/or ObR gene product activity, obR nucleic acid sequences can beutilized for the treatment of body weight disorders, including obesity.Where the cause of obesity is a defective ObR, treatment can beadministered, for example, in the form of gene replacement therapy.Specifically, one or more copies of a normal obR gene or a portion ofthe obR gene that directs the production of an obR gene productexhibiting normal function, can be inserted into the appropriate cellswithin a patient or animal subject, using vectors which include, but arenot limited to, adenovirus, adeno-associated virus, retrovirus, andherpes virus vectors, in addition to other particles that introduce DNAinto cells, such as liposomes.

Because the obR gene is expressed in the brain, including the choroidplexus and hypothalamus, such gene replacement therapy techniques shouldbe capable of delivering obR gene sequences to these cell types withinpatients. Thus, the techniques for delivery of the obR gene sequencesshould be designed to readily cross the blood-brain barrier. Thesetechniques are well known to those of skill in the art (see, e.g., PCTapplication, publication No. WO89/10134, which is incorporated herein byreference in its entirety), or, alternatively, should involve directadministration of such obR gene sequences to the site of the cells inwhich the obR gene sequences are to be expressed. Alternatively,targeted homologous recombination can be utilized to correct thedefective endogenous obR gene in the appropriate tissue; e.g., choroidplexus and/or hypothalamus. In animals, targeted homologousrecombination can be used to correct the defect in ES cells in order togenerate offspring with a corrected trait.

Additional methods that can be utilized to increase the overall level ofobR gene expression and/or ObR activity include the introduction ofappropriate ObR-expressing cells, preferably autologous cells, into apatient at positions and in numbers which are sufficient to amelioratethe symptoms of body weight disorders, including obesity. Such cells canbe either recombinant or non-recombinant. Among the cells that can beadministered to increase the overall level of obR gene expression in apatient are normal cells, preferably choroid plexus cells, orhypothalamic cells, which express the obR gene. The cells can beadministered at the anatomical site in the brain, or as part of a tissuegraft located at a different site in the body. Such cell-based genetherapy techniques are well known to those skilled in the art, see, forexample, Anderson, et al., U.S. Pat. No. 5,399,349; Mulligan & Wilson,U.S. Pat. No. 5,460,959.

Finally, compounds, identified in the assays described above, thatstimulate or enhance the signal transduced by activated ObR, forexample, by activating downstream signalling proteins in the ObRcascade, and thereby by-passing the defective ObR, can be used toachieve weight loss. The formulation and mode of administration willdepend upon the physico-chemical properties of the compound. Theadministration should include known techniques that allow for a crossingof the blood-brain barrier.

5.7. Pharmaceutical Preparations and Methods of Administration

The compounds that are determined to affect obR gene expression or ObRactivity can be administered to a patient at therapeutically effectivedoses to treat or ameliorate weight disorders, including obesity,cachexia, and anorexia. A therapeutically effective dose refers to thatamount of the compound sufficient to result in amelioration of symptomsof body weight disorders.

5.7.1. Effective Dose

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, for example, for determining the LD₅₀ (the dose lethal to 50%of the population) and the ED₅₀ (the dose therapeutically effective in50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects can be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography (HPLC).

5.7.2. Formulations and Use

Pharmaceutical compositions for use in accordance with the presentinvention can be formulated in a conventional manner using one or morephysiologically acceptable carriers or excipients.

Thus, the compounds and their physiologically acceptable salts andsolvates can be formulated for administration by inhalation orinsufflation (either through the mouth or the nose), or oral, buccal,parenteral, or rectal administration.

For oral administration, the pharmaceutical compositions can take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone, orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose, or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc, or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets can be coated by methods well known in the art. Liquidpreparations for oral administration can take the form of, for example,solutions, syrups or suspensions, or they can be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations can be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives, or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations can also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration can be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions can take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, for example, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, orother suitable gas. In the case of a pressurized aerosol, the dosageunit may be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, for example, gelatin for use in an inhaleror insufflator can be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds can be formulated for parenteral administration byinjection, for example, by bolus injection or continuous infusion.Formulations for injection can be presented in unit dosage form, forexample, in ampoules or in multi-dose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively, the active ingredient can be in powder form forconstitution with a suitable vehicle, for example, sterile pyrogen-freewater, before use.

The compounds can also be formulated in rectal compositions such assuppositories or retention enemas, for example, containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds canalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (e.g., subcutaneously orintramuscularly), or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials (e.g., as an emulsion in an acceptable oil), or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

The compositions can, if desired, be presented in a pack or dispenserdevice which can contain one or more unit dosage forms containing theactive ingredient. The pack can, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device can beaccompanied by instructions for administration.

6. EXAMPLE In Situ Localization of ObR

In the Example presented herein, it is demonstrated via binding studieswith Ob (leptin)-alkaline phosphatase (AP) fusion proteins that highaffinity Ob receptor is present in mammalian choroid plexus tissue. Itis further demonstrated that the fusion protein binding observed wasOb-specific, and not due to a non-specific alkaline phosphatase-basedartifact.

6.1. Materials and Methods

Construction and Expression of Ob-Alkaline Phosphatase (AP) FusionProteins. Two types of fusion protein were generated. Specifically,Ob-AP fusion proteins were generated in which the AP portion was at thecarboxyl terminus of the fusion protein, and AP-Ob fusion proteins weregenerated in which the AP portion was at the amino terminus of thefusion protein.

To produce mouse and human Ob-AP and AP-Ob fusion constructs, cDNAsequences were amplified by standard polymerase chain reaction (PCR)procedures. For mouse and human Ob-AP fusions, nucleotide sequencesencoding the entire open reading frames of mouse and human Ob,respectively were amplified from the corresponding cDNAs. Restrictionsites at the end of the amplification primers were cut with HindIII andBamHI (mouse) and inserted into the HindIII-BglII polylinker site ofAPtag-2, or BamHI and BglII (human) and inserted into the BglII site ofAPtag-2. For mouse and human AP-Ob fusion constructs, a new AP fusionvector expressing an AP molecule with its own signal peptide was firstgenerated (APtag-3) by replacing sequences between the HindIII and XhoIsites of APtag-2 with PCR amplified sequences of secreted placentalalkaline phosphatase (including signal sequence). A BglII site wasplaced so that fusions introduced into this site would be in-frame withthe AP protein. The sequences of the predicted mature forms of mouse andhuman Ob were then PCR amplified from the corresponding cDNAs.Restriction sites at the end of the amplification primers were cut withBamHI and BglII and inserted into the BglII site of APtag-3.

Each plasmid was transiently transfected into COS-7 cells (11.25 μg/150mm plate). Cells were grown to confluence and then media-conditioned for3 days. Cells were then centrifuged, 0.45 μm filtered, and stored at 4°C. with 20 mM Hepes (pH 7.0) and 0.05% sodium azide. Conditioned mediawere tested and quantitated for AP activity in a 96-well plate reader asdescribed by Flanagan and Leder (Flanagan, J. G. and Leder, P., 1990,Cell 63:185-194), except that homoarginine was omitted from all assays.

In Situ Fusion Protein Binding. Quartered mouse brains, isolated choroidplexus, cells, and cell lines were rinsed once with HBHA (Hank'sbalanced salt solution with 0.5 mg/ml BSA, 0.1% NaN₃, 20 mM HEPES [pH7.0]) in 12-well plates. Tissue was then incubated with tissue culturesupernatants containing AP-Ob fusion, Ob-AP fusion, or controlsupernatants (i.e., supernatants containing unfused AP only, containingAP-OB or OB-AP fusion proteins plus 80-fold molar excess of E.coli-derived recombinant OB, or supernatants from mock-transfected COScells), for 75 minutes with gentle rotation, at room temperature.Samples were then treated as described previously (Cheng, H. J. andFlanagan, J. G., 1994, Cell 79:157-168).

6.2. Results

To search for the Ob receptor, Ob-alkaline phosphatase fusion proteinswere constructed which would allow colorimetric detection of Ob binding.Specifically, cDNA molecules encoding the mouse and human Ob proteinswere inserted into the expression vectors APtag-2 and APtag-3, asdescribed, above, in Section 6.1. Insertion into the expression vectorAPtag-2 resulted in a fusion protein with Ob at the N-terminus of thefusion protein and placental alkaline phosphatase (AP) at theC-terminus. The resulting fusion protein is referred to as Ob-AP.Insertion into the vector APtag-3 resulted in fusion proteins with AP atthe N-terminus fused to the predicted mature form of the Ob protein atthe C-terminus. The resulting fusion protein is referred to as AP-Ob.Both forms of murine fusion proteins were secreted and both wereproduced at the predicted molecular weight of approximately 81 kDa.

Several strategies were employed in an effort to identify cells ortissues expressing the Ob receptor. Each of the cells, cell lines, andtissues tested as described herein were at least potentially involved inbody weight regulation. The first strategy employed was to attemptdirect binding assays with the Ob-AP and AP-Ob fusion proteins. Celllines examined by this strategy included the placental cell lines Be Wo(ATCC No. CCL98) and JAR (ATCC No. HTB144); the muscle cell lines L6(ATCC No. CRL1458) and BC3H (ATCC No. CRL1443); the neural cell linesPC12 (ATCC No. CRL1721) and NB41A3 (ATCC No. CCL147); the preadiposecell line 3T3-L1 (ATCC No. CRL173); and the liver cell line Hepa1-6(ATCC No. CRL1830). Also tested by this method were primary culturesfrom hypothalamus and primary cultures from cerebellum. None of thesestudies yielded positive binding results.

Second, attempts were made to identify cell lines expressing Ob receptorby examining changes in gene expression in response to the presence ofrecombinant Ob protein. The rationale being that changes in geneexpression, whether obR gene expression or the expression of genesfurther downstream in the Ob/ObR-related signal transduction pathway,would identify cells in which ObR was present.

This analysis was done by standard differential display analysis (seePardee et al., U.S. Pat. No. 5,262,311) of RNA derived from Ob-treatedor untreated cells. Briefly, RNA was isolated from cells which eitherhad or had not been exposed to Ob, and was amplified via RT-PCR in amanner which allowed a direct quantitative comparison of the levels ofindividual transcripts present in the RNA derived from the Ob-treatedcell lines relative to the Ob-untreated cell lines. Ob Cell lines testedby this approach were INS-1, 3T3-L1, Hepa1-6, L6, PC12, NB41A3 and BC3H.In addition, primary hypothalamic cultures were also tested. None of thecells tested exhibited a detectable quantitative difference inexpression pattern based on whether the cells had or had not beentreated with Ob.

Third, attempts to identify cells expressing Ob receptor were made bytreating cells with recombinant Ob protein and assaying for signs ofsignal transduction pathway activation. Specifically, cAMP changes weremonitored via ³H uptake, and tyrosine phosphorylation changes wereassayed via Western blots treated with anti-phosphotyrosine antibodies.Over twenty cell lines were examined in this manner. Specifically, thesecell lines included the mouse cell lines Y1 (adrenal cortex; ATCC No.CCL79), BC3H (smooth muscle-brain tumor; ATCC No. CRL1443), P19(embryonal carcinoma; ATCC No. CRL1825), 3T3L1 (preadipocyte; ATCC No.CRL173), Hepa1-6 (hepatoma; ATCC No. CRL1830), C2C12 (myoblast; ATCC No.CRL1772), NMUMG (mammary gland, normal epithelial; ATCC No. CRL1636),MM5MT (mammary gland; ATCC No. CRL1637), NB41A3 (neuroblastoma; ATCC No.CCL147), AtT20 (pituitary; ATCC No. CCL89), N MU LI (liver; ATCC No.CRL1638), BNL CL2 (liver; ATCC No. TIB73), and NCTC-1469 (liver; ATCCNo. CCL91); rat cell lines, including L6 (myoblast; ATCC No. CRL1458),PC12 (adrenal chromaffin; ATCC No. CRL1721), and H-4-II-E (hepatoma;ATCC No. CRL1548); and human cell lines, including SW872 (liposarcoma;ATCC No. HTB92), Hepa G2 (liver; ATCC No. HB8065), and neuroblastomacell lines, including SK-N-SH (ATCC No. HTB11). Again, no Ob-dependentdifferences were observed in any of the cells tested.

After an extensive search of mammalian cell lines and tissues, adultmouse brains were quartered, treated with AP-Ob fusion protein, washed,and tested for bound AP activity of the fusion protein usinghistological techniques, as described, above, in Section 6.1.Reproducible binding of the AP-Ob fusion protein was observed in therodent brain choroid plexus (within the lateral and third brainventricals). No AP-Ob staining was observed, however, in the braintissues surrounding the choroid plexus. The choroid plexus is a tissuelargely responsible for the generation of the cerebral spinal fluid.Further, choroid plexus tissue is considered to be one of the“guardians” of the blood-brain barrier.

Control AP staining was performed on tissues treated with unfused AP andon tissues that had been treated with AP-Ob in the presence of an excessof unfused Ob added to compete for the binding of the fusion protein.Staining similar to that observed for the Ab-Ob fusion protein was notobserved in either of these controls, demonstrating that the AP-Obbinding observed was Ob-specific, and not due to an AP-based artifact.

In summary, therefore, only after employing several strategies, was acell surface molecule which binds Ob located; and this cell surfacemolecule was found within a specific region of the brain, the choroidplexus.

7. EXAMPLE Cloning of the Murine ObR Gene

Described, below, in Section 7.2.1, is the successful cloning of a shortform Ob receptor cDNA, famj5312, from expression libraries constructedusing murine choroid plexus RNA. The expression libraries were screenedusing AP-Ob fusion protein binding, as described, above, in the Examplepresented in Section 6. Section 7.2.2, below, describes the nucleotidesequence of the short form Ob receptor coding region and, further,describes the amino acid sequence of the Ob short form receptor protein.Section 7.2.3, below, describes competitive binding studiesdemonstrating that the protein encoded by the isolated cDNA encodes areceptor exhibiting high affinity binding for both mouse and human Obprotein. Section 7.2.4 describes studies that verify the authenticity ofthe isolated obR cDNA clone.

The high affinity Ob binding exhibited by the ObR, coupled with itshomology to the Class I family of cytokine receptors, as described,below, indicates that the ObR is involved in the control of mammalianbody weight, via signal transduction triggered by its binding to Obligand.

7.1. Materials and Methods

Choroid Plexus mRNA Isolation. Total RNA was isolated from 300 mousechoroid plexuses in batches of 100, using the guanidiniumisothiocyanate/CsCl method of Chirgwin et al. (1979, Biochemistry18:5294) as described by R. Selden In Current Protocols for MolecularBiology (4.2.3 Supplement 14). After quantitation, the RNA was dilutedto 1 mg/ml in distilled, deionized water and incubated for 30 minutes at37° C. with an equal volume of DNase solution (20 mM MgCl₂, 2 mM DTT,0.1 units DNase, 0.6 units RNase inhibitor in TE) to removecontaminating DNA. The RNA was extracted with phenol/chloroform/isoamylalcohol, and ethanol precipitated. After quantitation at 260 nm, analiquot was electrophoresed to check the integrity of the RNA. A totalof 320 μg of total RNA was purified.

Poly A⁺ RNA was isolated using an Oligotex-dT kit (catalog # 70042) fromQiagen (Chatsworth, Calif.) as described by the manufacturer. Afterquantitation, the mRNA was ethanol precipitated and resuspended at 1mg/ml in distilled, deionized, DEPC-treated water. A total of 11 μg ofpoly A⁺ RNA was purified.

Library Construction. cDNA was synthesized according to the method ofGubler and Hoffman (Gene 25:263, 1983) using a Superscript Plasmid cDNAsynthesis kit (Catalog # Series 8248) purchased from Life Technologies(Gaithersburg, Md.). The cDNA obtained was ligated into the NotI/SalIsites of the mammalian expression vector pMET7, a modified version ofpME18S, which utilizes the SRα promoter as described previously (Takebe,Y. et al., 1988, Mol. Cel. Biol. 8:466). This vector was chosen becauseit contains a strong eukaryotic promoter, is expressed in COS7 cells,contains the ampicillin resistance gene, and is only 3.0 kb in length.The small size of the vector is important because it increases theprobability of cloning large cDNAs. Other comparable vectors are 4.8 kband larger, thereby increasing the chances of imperfect replication, andreducing the probability of cloning large cDNAs. Ligated cDNA wasethanol precipitated and resuspended in distilled, deionized,DEPC-treated water at 25 ng/ml. One μl of the DNA was transformed byelectroporation per 40 μl of electrocompetent DH10B E. coli in a 0.1 cmcuvette.

cDNA was synthesized twice and used to construct two independent mousechoroid plexus libraries: mCP (mouse choroid plexus) A and mCP D.

DNA Preparation. Based on titers of the cDNA transformations,96-deepwell plates were inoculated with 150 cfu/well of primarytransformants in 1 ml of Luria broth containing ampicillin (LB-amp).Primary transformants that were grown only 1 hour at 37° C. prior toaliquoting were used to avoid the overgrowth of smaller insert clonesand thus under representation of larger clones in the 150 cfu pools.Cultures were grown 15-16 hours at 37° C. with aeration. Prior toprepping, 100 μl of cell suspension was removed and added to 100 μl of50% glycerol, mixed, and stored at −80° C. (glycerol freeze plate).

DNA was prepared using the Wizard™ Minipreps DNA Purification Systems(Promega, Madison, Wis.; Catalog No. A7100) employing modifications fora 96-well format. The protocol was as follows:

-   -   1) Cultures were centrifuged in 96-deepwell plates at 3200 rpm,        for 10 minutes, at 4° C., and the supernatants were removed.    -   2) 140 μl each of cell resuspension solution (50 mM Tris-HCl (pH        7.5), 10 mM EDTA, 100 μg/ml RNase A), cell lysis solution (0.2 M        NaOH; 1.0% SDS) and neutralization solution (1.32 M Potassium        acetate, pH 4.8) were added, in order, with vortexing 1.4        seconds after addition of each reagent, to ensure good mixing.    -   3) Plates were placed in ice water for 15 minutes.    -   4) Samples were centrifuged at 3200 rpm, for 10 minutes, at 4°        C.    -   5) Supernatants were transferred to 96-well Polyfiltronics        polypropylene filterplate (10 micron, 0.8 ml).    -   6) 500 μl WP resin were added and incubated 3-5 minutes at room        temperature; suction was applied to the plate.    -   7) Samples were washed three times with 640 μl of the        resuspension solution.    -   8) Samples were centrifuged at 3200 rpm, for 5 minutes, at room        temperature, to remove residual buffer.    -   9) Samples were eluted 2-5 minutes with 40 μl room temperature        water.    -   10) Eluted DNA was centrifuged through to microwell plates at        3200 rpm, for 5 minutes, at room temperature.    -   11) DNA was quantitated.

Pooling Strategy. The pooling strategy was devised to provide optimalsized pools, 1200 cfu, for transfection and detection, and quickbreakdown to the smaller pools of 150. Once a positive pool of 150 wasidentified, between 400 to 800 individual clones were needed to providerepresentation of the pool. Using a single pool of 1200 cfu initiallywould have meant fewer DNA probes but would have required the use ofmore individual clones (3200-6400) in the final identification step,thereby requiring significantly more time to identify a positive clone.

DNAs totalling 5 μg were pooled equally from eight wells, one column, togive a total of 1200 cfu. Thus, each 96-well plate gave rise to 12pooled DNAs for transfection into COS-7 cells.

When a positive pool was identified, DNA was prepared from each of theeight wells constituting the pool and retransfected into COS-7 cells.When a positive well was identified, the well was broken down by platingout an aliquot of the glycerol freeze of that well such that severalthousand individual colonies were obtained. For each positive well,between 400 and 800 colonies were picked and arrayed in a 96-wellformat, DNA was obtained, as described above, and the DNA from 24 wellswas pooled for transfection. DNA representing each individual clone froma positive row was isolated and transfected for final identification.

Quantitative Ob cell surface binding analysis. Quantitative cell surfacebinding assays with AP-Ob fusion proteins were performed essentially asdescribed previously for Kit-AP (Flanagan, J. G. and Leder, P., 1990,Cell 63:185-194).

Ob Protein. The recombinant murine Ob protein used herein has beendescribed previously (Campfield et al., 1995, Science 269:546-549). Therecombinant human Ob protein used herein was purified from Baculovirussupernatants with a monoclonal antibody column containing monoclonalantibody directed against human Ob. The purified recombinant human Obprotein was judged by standard Coomasie blue staining to be greater than95% pure.

DNA Sequencing. Sequencing and sequence assembly were performed asdescribed previously (International Polycystic Kidney Consortium, 1995,Cell 81:289-298).

Northern Analysis. Northern blot analysis of poly A+ mRNA from varioustissues (Clontech) was probed, using standard techniques (Chirgwin, J.M. et al. 1979, Biochemistry 18:5294-5299), with labeled DNA amplifiedfrom sequences encoding the murine ObR extracellular domain.

RT-PCR. Reverse transcription PCR (RT-PCR) reactions were performed on 1μg total RNA utilizing standard techniques (Zhang, Y. et al., 1994,Nature 372:425-432). Specifically, first strand cDNA was prepared usingrandom hexamers. The first strand cDNA was then PCR amplified usingprimers derived from sequences encoding the ObR extracellular domain orG3PDH control primers.

7.2. Results 7.2.1. Cloning of the OB Receptor from Mouse Choroid Plexus

The strong, Ob-specific binding of the AP-Ob fusion protein to themurine choroid plexus described above, in the Example presented inSection 6, suggested that an Ob receptor could be expressed at highlevels within this tissue. In order to attempt to clone a cDNA encodingthe Ob receptor, therefore, the choroid plexuses from 300 mice weredissected, and a total of 11 μg poly A⁺ RNA was isolated from the tissueto be used to construct cDNA libraries as described above, in Section7.1.

Initially, 3 μg poly A⁺ were used to generate cDNA, to be used inconstructing mouse choroid plexus cDNA library A (mCP A). All of thecDNAs generated that were greater than 500 bp in size (261 ng) werepooled and 90 ng were ligated to pMET7. Transformation of this ligatedcDNA into electrocompetent DH10B E. coli resulted in a library ofapproximately 7.2×10⁵ cfu, with an average size of 1 kb.

Recognizing that cDNA library A did not contain a sufficient number ofclones containing inserts large enough to encode a receptor at astatistically reasonable frequency, a second 3 μg of poly A⁺ RNA wasused to generate 758 ng of cDNA. 32 ng of cDNA representing the largesttwo fractions of cDNA were pooled and ligated into pMET7. Transformationof these ligated cDNA molecules resulted in mouse choroid plexus libraryD (mCP D), with 2.4×10⁵ cfu and an average insert size of 2 kb. Usingonly the largest two fractions of cDNA ensured that the library would bebiased towards large cDNAs. This was confirmed by characterizing theinsert sizes of ten clones; seven clones had inserts greater than 2 kbin length and no clones were seen with inserts smaller than 1 kb. Thiswas in contrast to the library A where 16 out of 20 clones were smallerthan 1 kb.

DNA representing 6×10⁵ cfu (40 plates) was prepared and pooled from themouse choroid plexus library A. DNA representing 2.4×10⁵ cfu (16 plates)was prepared from mouse choroid plexus library D.

For screening purposes, the libraries were produced as pools of 150clones, with a mixture of 8 pools being used in each transfection (i.e.,1200 clones/transfection). Pooled DNA was transiently transfected intoCOS-7 cells, and the cells were screened by incubation with supernatantscontaining the murine AP-Ob fusion protein, washed, and stained for APactivity in situ, all as described, above, in Sections 6.1 and 6.2. Oncea positive pool was identified, the 8 individual subpools were eachtested separately, and the resulting positive subpool was furthersubdivided until a single positive clone was identified.

A total of 632 DNA pools were derived from libraries A and D, with atotal of 10 independent positive pools being identified. All of thesepositive pools were successfully broken down into subpools of 150 cloneseach, and one positive subpool was further subdivided until a singlepositive clone was identified. The clone, which contained a 5.1 kB cDNAinsert, was designated famj5312.

7.2.2. The Ob Receptor (ObR) and ObR Gene

The famj5312 murine obR cDNA clone isolated, as desexibed above, inSection 7.2.1, contained an insert of approximately 5.1 kb. Thenucleotide sequence obtained from this clone is depicted in FIGS. 1A-1D(SEQ ID NO:1). The nucleotide sequence of the clone revealed a singleopen reading frame, the ObR derived amino acid sequence of which is alsodepicted in FIGS. 1A-1D (SEQ ID NO:2).

The deduced 894 amino acid sequence of the murine ObR protein beginswith a methionine whose codon is within a DNA sequence that isconsistent with a translation initiation site. The ObR amino acidsequence begins with a hydrophobic signal sequence from amino acidresidues 1-23, which is typical of proteins that are to be eithermembrane-associated or secreted.

The murine Ob receptor protein contains a single hydrophobictransmembrane domain from amino acid residues 838-860, indicating thatthe Ob receptor spans the cell membrane once.

The position of the transmembrane domain indicates that theextracellular portion of the mature murine ObR protein spans from aminoacid residue 24 to amino acid residue 837. A database search revealsthat the extracellular domain of ObR contains regions of homology whichplace ObR into the Class I family of cytokine receptors (for reviews,see, e.g., Heldin, C.-H., 1995, Cell 80:213-223; and Kishimoto, T. andTetsuya, T., 1994, Cell 76:253-252). ObR appears to be most closelyrelated to the gp130 signal transducing component of the IL-6 receptor,the GSF receptor and the LIF receptor. Alignment studies of ObR andgp130 amino acid sequences revealed that, although the overall sequenceidentity between the two proteins is low, the characteristic conservedcysteine residues, the Trp-Ser-X-Trp-Ser motif, and other amino acidresidues conserved within the class I family of proteins are clearlyevident.

Following the single transmembrane domain, the murine Obr proteincontains a short cytoplasmic domain of 34 amino acids (i.e., amino acidresidues 861-894). Homology comparisons also reveal that the firsttwenty three amino acids of the ObR cytoplasmic domain show a 30%identity to membrane proximal sequences of the LIF receptor. Reversetranscription PCR amplification of obR mRNA from total RNA confirmed thepresence of obR transcript (a single band of about 5 kb) in choroidplexus, and also demonstrated its presence in hypothalamus. Further,Northern blot analysis of poly A⁺ RNA derived from several mouse tissuesrevealed that obR mRNA is present in additional tissues, such as lungand kidney.

7.2.3. The OB Receptor Strongly Binds OB Protein

An analysis of the binding of AP-Ob to the ObR encoded by the obR cDNAdescribed above. in Section 7.2.2, was conducted. The results of thisanalysis, depicted in FIGS. 2A-2B, demonstrate that the ObR exhibitsstrong, Ob-specific binding to both mouse and human Ob protein.

A quantitative analysis of the binding of the AP fusion proteins isshown in FIGS. 2A-2B. After transient cransfection of the ObR clone intoCOS cells, strong binding of 1 nM murine AP-Ob is detected (relative tomock transfected COS cells or ObR transfected COS cells incubated withunfused AP) (FIG. 2A). This binding is nearly completely inhibited by100 nM untagged recombinant mouse or human leptin protein, demonstratingthat this receptor can bind native Ob. A fusion between AP and human Obalso binds mouse ObR with high affinity, as does a fusion protein withmouse leptin at the N-terminus and AP at the C-terminus (Ob-AP).Scatchard analysis of the binding of mouse AP-Ob (FIG. 2B) produced avalue for the dissociation constant (K_(D)) of 0.7×10⁻⁹ M.

7.2.4. Authenticity of the famj5312 Clone

The authenticity of the isolated obR famj5312 clone was tested inseveral ways. First, 8 independently isolated clones (in subpools of 150clones each) were PCR amplified with primers made to obR sequences 3′ ofthe stop codon. Sequencing verified that all 8 clones contained the same3′ untranslated sequences. In addition, the regions of 5 independentlyisolated clones encoding the ObR C-terminus were sequenced and each wasshown to utilize the same stop codon. Finally, reverse transcription PCR(rt-PCR) of choroid plexus total RNA isolated from a different mousestrain (C57/BLKsJ) than that from which the cDNA libraries were derivedgenerated an identical PCR product containing a stop codon in the samelocation. These data indicated that the isolated famj5312 cDNA clone wasneither a chimeric clone nor was it the result of a rare aberrantsplicing event, but, rather, represents a clone which encodes thepredominant form of the ObR receptor in the choroid plexus.

7.2.5. Cloning Mouse Long Form ObR Encoding Nucleic Acids

As described herein, we have cloned the murine ObR long form.

In order to find the mouse homolog of the human long form of the obRgene (FIGS. 3A-3F), semi-nested PCR was performed on first strand cDNAisolated from mouse hypothalamus, Ks, and choroid plexus, db and Ks,with 5′ primers from the region just before mouse short form starts todiverge from the human long form, and 3′ degenerate primers designedfrom the human ObR homolog intracellular region: The complete transcriptwas further characterized by 3′ RACE.

Total mRNA was prepared from C57Bl/KS (KS) and C57B1/KS-db (db) choroidplexus and hypothalamus. cDNA was reverse-transcribed from 1 μg of cDNAof mRNA using random hexamer or oligo dT as primer with SuperscriptReverse Transcriptase from GIBCO-BRL. A total 24 μg of cDNA was made.For PCR, cDNA was diluted 1:200 and 3 μg of the diluted cDNA was used ina 25 μl reaction.

The first round of PCR reactions used a 5′ primer encoding the mouse ObRprotein sequence PNPKNCSW (SEQ ID NO:29), and consisting of nucleotides5′-CCAAACCCCAAGAATTGTTCCTGG-3′ (SEQ ID NO:30), and a reverse degenerateprimer complementary to the nucleotide sequence encoding KIMENKMCD (SEQID NO:31), adjacent to the carboxy terminus of the human long form andconsisting of nucleotides 5′-TC(GA)CACAT(CT)TT(GA)TT(GATC)CCCATTATCTT-3′(SEQ ID NO:32).

For the second round of PCR reactions, the 3′ primer was the same, andthe 5′ primer, which was internal to the previous 5′ primer, encoded themouse ObR protein sequence AQGLNFQK (SEQ ID NO:33), and consisted ofnucleotides 5′-GCACAAGGACTGAATTTCCAAAAG-3′ (SEQ ID NO:34).

PCR reactions were carried out as described above, except the nested PCRprofile was 94° C. for 3 minutes; 94° C. for 30 seconds, 57° for 30seconds, 72° C. for 40 seconds for 30 cycles; 72° C. for 5 minutes forone cycle.

DNA sequencing was performed on the automatic ABI 373A and 377 DNAsequencer by using the Taq cycle™ sequencing kit (Applied Biosystems,Foster City, Calif.). Sequence analysis was performed using Sequencher.

Semi-nested PCR of the nucleic acids encoding the intracellular domainof murine long form ObR was also performed on mRNA isolated fromhypothalamus in order to obtain sufficient quantifies of a specific PCRproduct encoding the mouse long form of obR gene. Sequencing of the PCRproduct (FIGS. 6A-6F) confirmed that this DNA encodes the mouse homologof the long form of ObR. The transcripts of the short and long forms areidentical until the fifth codon 5′ of the stop codon of the short formand then diverge completely, suggestive of alternative splicing. Thededuced amino acid sequences from mouse long form and the human ObR arehomologous throughout the length of the coding region and share 75%identity (FIGS. 7A-7B).

7.2.6. Expression Profile of ObR mRNA

As a first step in understanding the tissue distribution of ObR, theexpression of its mRNA was examined in various murine tissues. To thisend, Northern blot analysis of poly A⁺ mRNA (2 μg/lane) obtained fromvarious mouse tissues (heart, brain, spleen, lung, liver, skeletalmuscle, kidney, and testes; Clontech, Palo Alto, Calif.) was probed withlabelled DNA amplified from sequences encoding the ObR extracellulardomain. Hybridizations were done in Rapid-hyb™ buffer (Amersham) at 65°C. following the manufacturer's instructions.

In most tissues, the obR mRNA appears as a single band, slightly largerthan 5 kb, indicating that the 5.1 kb cDNA clones described herein arefull-length. Of the tissues assayed, expression was seen in lung,kidney, and total brain. No expression was detected in testes.

RT-PCR amplification of the obR mRNA from total RNA confirmed thepresence of this transcript in choroid plexus and also demonstrated itspresence in hypothalamus. The RT-PCR reactions were performed on 1 μgtotal RNA isolated from mouse choroid plexus or hypothalamus. Tissueswere isolated from db/db mice (C57Bl/BLKsJ background) or +/+littermatecontrols. First strand cDNA, prepared using random hexamers, was PCRamplified using primers derived from sequences encoding the ObRextracellular domain or G3PDH control primers. No bands were detectedfrom the amplification of mock reverse-transcribed total RNA controlsrun in parallel.

8. EXAMPLE The obR Gene is the db Gene

The experiments and studies described below demonstrate that the obRgene maps to the db locus, and that the obR gene in db mice is a mutantform of obR that results in transcription of an aberrantly spliced mRNAhaving a 106 nucleotide insert resulting in a truncated long form murineObR protein that is identical to murine short form ObR.

8.1. The obR Gene Maps within the db Genetic Interval

In the Example presented herein, studies are described which indicatethat the obR gene maps to a 4 to 5 cM region on mouse chromosome 4 whichrepresents the same region to which the db locus maps.

8.1.1. Materials and Methods

PCR Amplification. The following famj5312-derived primers were used foramplification of mouse genomic DNA: forward primer,5′-GCTGCACTTAACCTGGC-3′ (SEQ ID NO:23); reverse primer,5′-GGATAACTCAGGAACG-3′(SEQ ID NO:24).

The PCR reaction mixture contained 6 μl of template DNA (10 ng/μl), 1.4μl 10× Perkin Elmer (Norwalk, Conn.) PCR buffer, 1.12 μl dNTPs (2.5 mM),1.05 μl Forward primer (6.6 μM), 1.05 μl Reverse primer (6.6 μM), 0.38μl H₂O and 3 μl AmpliTaq Hotstart™ polymerase (Perkin Elmer; 0.5 U/μl).

The amplification profile was as follows: 94° C., 2 minutes, at whichpoint the ampliTaq was added, then 30 cycles of 94° C. for 40 seconds,55° C. for 50 seconds, and 72° C. for 30 seconds.

A second set of primers were utilized under the same conditions exceptthat the 55° C. cycle was conducted at 52° C.: forward primer,5′-CACTATTTGCCCTTCAG-3′(SEQ ID NO:25); reverse primer,5′-GCCTGAGATAGGGGTGC-3′(SEQ ID NO:26).

Electrophoresis. Samples were run on both nondenaturing 8% acrylamidegels run at 45 W, room temperature, for 3 hours and nondenaturing 10%acrylamide SSCP (single stranded conformational polymorphism) gels runat 20 W, 4° C., for 2.5 hours.

Both types of gels were stained with SYBR Green I and scanned on an MDFluorimager™ Both types of gels gave interpretable results.

8.1.2. Mapping of the famj5312 obR cDNA Clone

PCR primers were designed from the coding sequence of famj5312 cDNA, asdescribed in Section 8.1. These primers amplified a 192 bp fragment fromC57Bl/6J genomic DNA, consistent with the base pair length between thetwo primers in the obR cDNA, and a 195 bp fragment from the wild-typederived Mus spretus strain SPRET/Ei. The 3 bp insertion in the Musspretus allele codes for an additional Asn between amino acid residues45 and 46. The genetic segregation of the Mus spretus 195 pb allele ofObR was followed in 182 backcross progeny of the cross (C57Bl/6J×Musspretus) F₁ females×C57Bl/6J males by both Single StrandedConformational Polymorphism (SSCP) gel electrophesis and nondenaturinggel electrophoresis for size determination. The segregation pattern ofthe Mus spretus allele was compared to the segregation pattern of 226other genetic loci that have been mapped in this backcross panel. Byminimizing the number of multiple crossovers between obR and othermarkers, it was determined that obR maps to murine chromosome 4,approximately 2.2±1.6 cM distal to the marker D4Mit9 and 4.6±1.6 cMproximal of the marker D4Mit46. The genetic map position of obR wasfurther refined by mapping additional genetic markers. The obR gene maps0.6±0.6 cM distal from D4Mit255 and 0.6±0.6 cM proximal of D4Mit155; seeFIG. 8.

Additional primer pairs were designed (forward=5′-CACTATTTGCCCTTCAG-3′(SEQ ID NO:27); reverse=5′-GCCTGAGATAGGGGTGC-3′ (SEQ ID NO:28)) from the3′ sequence of famj5312 cDNA, which also revealed a polymorphism on SSCPgels between C57Bl/6J genomic DNA and that of the wild derived Musspretus strain SPRET/Ei. Again this permitted the genetic mapping offamj5312 cDNA, now using a different fragment of the clone. The mappingof this polymorphism was 100% concordant with the mapping of famj5312reported above, both confirming the mapping of obR and indicating thatthe famj5312 cDNA clone was not chimeric.

8.1.3. Definition of the Murine db Genetic Region

The mouse db gene was originally mapped to mouse chromosome 4 (Hummel,K.-P. et al., 1966, Science 153:1127-1128). This genetic localizationhas been refined (Bahary et al., 1990, Proc. Natl. Acad. Sci. USA87:8642-8646; Bahary et al., 1993, Genomics 16:113-122) to place dbwithin a genetic interval of 1.5 cM between the proximal Ornithinedecarboxylase 4 (Odc4) locus and the anonymous distal markers D4Rck22and D4Rck69. Bahary et al. 1993, supra, also report D4Mit205 as being1.1 cM proximal to Odc4. Hence, relative to D4Mit205, the db gene wasmapped approximately 2.2 cM distal.

The db allele originally arose on the C57Bl/BLKsJ inbred strain. The dbmutation has subsequently been transferred to other genetic backgroundsto form congenic strains. By typing animals of the congenic strainC57Bl/6J-m db, it was possible to define the genetic interval withinwhich the db gene had to reside on mouse chromosome 4. By this analysis,the interval that must contain the db gene was defined as theapproximate 4 cM between the proximal anonymous DNA marker D4Mit255 andthe distal markers D4Mit331 and D4Mit31. (Genetic distance as defined onthe Mit map; Dieteich et al., 1994, Nature Genetics 7:220-245; Copelandet al., 1993, Science 262:67; Whitehead Institute/MIT Center for GenomeResearch, Genetic Map of the Mouse, Database Release 10, Apr. 28, 1995).It should be noted that the interval defined by Bahary et al. 1993,supra, appears to be a few centimorgans proximal of the region asdefined herein. See FIG. 8, in which the distance between D4Mit255 andD4Mit31 is about 5.1 cm.

By comparing the mapping data for famj5312 with the db mapping datadescribed above, the map position of famj5312, 0.6±0.6 cM distal fromD4Mit255 and 0.6±0.6 cM proximal of D4Mit155, is in complete accordancewith obR being the db gene.

8.2. The obR Mutation in db Mice Results in a Truncated Long FormReceptor 8.2.1. Materials and Methods

Total mRNA was prepared from C57Bl/KS (KS) and C57Bl/KS-db (db) choroidplexus and hypothalamus. cDNA was reverse-transcribed from 1 μg of cDNAof mRNA using random hexamer or oligo-dT as primers with Superscript™Reverse Transcriptase from GIBCO-BRL. A total 24 μg of cDNA was made.For PCR, cDNA was diluted 1:200 and 3 μg of the diluted cDNA was used ina 25 μl reaction.

From the mouse short form cDNA clone, famj5312, and the long form cDNAclone (FIGS. 6A-6F), primers were designed covering the entire codingregion of both the short and long forms of obR cDNA. Overlapping PCRfragments with an average size of 600 bp were generated from eachsample. PCR products were electrophoresed on an 0.8% low melting agarosegel. DNA was isolated from the gel and agarased. Agarased DNA fragmentswere sequenced with both end primers as well as internal primers.

PCR Conditions. The 25 μl PCR reaction contained 2 mM MgCl₂, 0.5 mM ofeach primer, 200 mM each of dATP, dTTP, dCTP and dGTP, and 0.5 units ofTaq polymerase in 1× Taq polymerase buffer (Perkin-Elmer). All PCRreactions were performed in the GeneAmp™ PCR System 9600 (Perkin-Elmer).Unless otherwise described, the general PCR profile was 94° C. for 3minutes; 94° C. for 10 seconds, 57° C. for 10 seconds, 72° C. for 40seconds for 35 cycles; and 72° C. for 5 minutes, for one cycle.

DNA sequencing and Sequence Analysis. DNA sequencing was performed onthe automatic ABI 373A and 377 DNA sequencer by using the Taq cyclesequencing kit (Applied Biosystems, Foster City, Calif.). Sequenceanalysis was performed using Sequencher.

8.2.2. Results

Semi-nested PCR was performed on mRNA isolated from choroid plexuses ofKS and db mice. The PCR product generated using the db cDNA as templatewas approximately 100 bp longer than that using Ks DNA as template. ThePCR products from both were directly sequenced. No sequence differencewas detected within the coding sequence of the short form of the mRNAspecies expressed in the choroid plexus of these mice. However, upon thesequencing of the PCR product that was generated starting from thetransmembrane domain shared by the two forms and ending in theintracellular domain specific for the long form, a difference becameapparent between db/db and control in several tissues. The sequencingdata showed that the putative db long form of obR has an additional 106bp insertion in the normal long form transcript (FIG. 9). This 106 bpincludes sequence encoding the last five amino acids, stop codon as wellas 88 bp 3′ UTR region of the short form. The db long form produces atruncated ObR protein identical to the short form which lacks theintracellular domain. The normal long form was not detected in any dbtissues, nor was the db long form detected in control tissues.

To understand the mechanism of this apparent splicing error, the obRgenomic sequences of the db/db and control mice were compared. A singlenucleotide change of G→T was discovered 2 bp immediately after the 106bp insertion site in db/db mice. This change creates a splice donorwhich converts the 106 bp fragment to an exon inserted in the db longform. Because of this insertion, the db long form produces only atruncated protein, which does not have the intracellular signal domain.Since the class I cytokine receptors to which ObR is most closelyrelated all have a long intracellular domain, the long intracellulardomain of the long form is crucial for initiating intracellular signaltransduction. These data support the role of this receptor in weightmodulation, and the failure to produce ObR long form as the cause of thesevere obese phenotype in db/db mice.

9. EXAMPLE Cloning Human ObR Encoding Nucleic Acids

Described herein is the cloning and identification of cDNA and gemonicDNA which encode human obR.

9.1. Cloning the Human Obr cDNA

The famj5312 cDNA insert was used to probe a human fetal brain cDNAlibrary in the Uni-Zap XR™ vector obtained from Sratagene (La Jolla,Calif.). A cDNA library generated from a human fetal brain was chosenbecause of the likelihood that this library would contain cDNAs presentin the entire brain, including the choroid plexus, the tissue source ofthe mouse obR cDNA, as well as cDNAs present in the hypothalamus.

The cDNA library was plated on 20 plates with approximately 50,000pfu/plate. Duplicated filter lifts were done on each plate with AmershamHybond-N™ nylon membrane filters. The filters were denatured,neutralized, and cross-linked according to standard procedures. Theprobe was radioactively labelled by random priming in the presence of³²P labelled nucleotide. The filters were hybridized with probeovernight at 65° C. in Church's buffer (7% SDS, 250 mM NaHPO₄, 2 μMEDTA, 1% BSA). The next day, filters were washed in 2×SSC/0.1% SDS for20 minutes at 65° C., then in 0.1×SSC/0.1% SDS for 10 minutes. They werethen exposed to Kodak film at −80° C. for 5 hours.

After matching duplicated filters, 13 duplicated signals were observed.Secondary plating was followed by plating out 10 μl of 1:1000 dilutionof each primary plug. The same probe, hybridization and wash conditionswere used as above. Film was exposed at 80° C. for 2 hours. Only 1 ofthe 13 original positives produced a duplicate signal on the film.

Four independent plaques from the positive plate were processed andexcised with ExAssist™ helper phage, XL1-Blue cells and SOLR cells, asdescribed by Stratagene. Excision products were then plated out onLB/Amp plates and incubated at 37° C. overnight. One white colony waspicked up from each plate and grown in liquid LB/Amp at 37° C.overnight. The next day, mini-preps were done with the Promega Wizard™Mini-prep kit. The sizes of the inserts were determined by digesting themini-prep products with EcoRI and XhoI. One of the four clones (d) hadan insert of approximately 6 kb.

DNA for sequencing was prepared using a Qiagen Plasmid Maxi kit.

FIGS. 3A-3F depict the nucleodde sequence (SEQ ID NO:3) of human obRcDNA encoding the signal sequence (amino acid residue 1 to about aminoacid residue 20), extracellular domain (from about amino acid residue 21to about amino acid residue 839), transmembrane domain (from about aminoacid residue 840 to about amino acid residue 862), and cytoplasinicdomain (from about amino acid residue 863 to about amino acid residue1165).

9.2. Cloning Human obR Genomic DNA

As described herein, human obR genomic DNA has been cloned.

The famj5312 cDNA insert was used to probe human high density PACfilters purchased from Genome Systems Inc. (Catalog No. FPAC-3386). Theprobe was random prime labelled using the Prime-It™ kit (Stratagene;Catalog No. 300392). The hybridization was carried out in AmershamRapid-hyb™ buffer according to the manufacturer's recommendations. Thefilters were then washed in 2×SSC/1% SDS at 65° C. and exposed to Kodakfilm at −80° C.

Eleven putative positive PAC clones were identified. Their grid positionwas determined, and the clones were purchased from Genome Systems, Inc.

The clone at grid position P298-K6, which we have designated hobr-p87,was further validated as containing the entire ObR coding region by PCRtesting with primer pairs from the 5′(obRF4 and obRR4) and 3′(obRS andobRO) ends of the obR open reading frame. The primers used in thisvalidation were as follows:

(SEQ ID NO:35) obRF4: 5′-CTGCCTGAAGTGTTAGAAGA-3′; (SEQ ID NO:36) obRR4:5′-GCTGAACTGACATTAGAGGTG-3′; (SEQ ID NO:37) obRS:5′-ACCTATGAGGACGAAAGCCACAGAC-3′; (SEQ ID NO:38) obRO:5′-TGTGAGCAACTGTCCTCGAGAACT-3′.

The hobr-p87 clone was deposited with the ATCC on Dec. 28, 1995.

10. EXAMPLE Construction of ObR Immunoglobulin Fusion Proteins 10.1.Preparation of OBR-IG Fusion Proteins

The extracellular portion of human ObR is prepared as a fusion proteincoupled to an immunoglobulin constant region. The immunoglobulinconstant region may contain genetic modifications including those whichreduce or eliminate effector activity inherent in the immunoglobulinstructure. (See, e.g., PCT Publication No. WO88/07089, published Sep.22, 1988). Briefly, PCR overlap extension is applied to join DNAencoding the extracellular portion of human ObR to DNA encoding thehinge, CH2 and CH3 regions of human IgG1. This is accomplished asdescribed in the following subsections.

10.2. Preparation of Gene Fusions

PCR reactions are prepared in 100 μl final volume composed of Pfupolymerase and buffer (Stratagene) containing primers (1 μM each), dNTPs(200 μM each), and 1 ng of template DNA.

DNA fragments corresponding to the DNA sequences encoding the ObR ECD,or a portion thereof that binds Ob, are prepared by polymerase chainreaction (PCR) using primer pairs designed so as to amplify sequencesencoding the entire human ObR ECD as well as a small amount of 5′noncoding sequence. For example, the forward primer:5′-GTCACGATGTCGACGTGTACTTCTCTGAAGTAAGATGATTTG-3′ (SEQ ID NO:39)corresponds to nucleotides −20 to +8 in FIGS. 3A-3F with an additional14 nucleotides (containing a SalI site) at the 5′ terminus. The reverseprimer: 5′-GTCAGGTCAGAAAAGCTTATCACTCTGTGTTTTTCAATATCATCTTGAGTGAA-3′ (SEQID NO:40) corresponds to the complement of nucleotides +2482 to +2517 inFIGS. 3A-3F, with an additional 18 nucleotides (containing a HindIIIsite) at the 5′ terminus. A cDNA encoding human ObR serves as thetemplate for amplifying the extracellular domain. PCR amplification withthese primers generates a DNA fragment that encodes ObR extrarellulardomain.

In a second PCR reaction, a second set of primers are designed toamplify the IgG constant region (i.e., the hinge, CH2, and CH3, domains)such that the reverse primer has an unique restriction site and thesequence of the forward primer has a 5′ terminus that is complementaryto the 5′ terminal region of the reverse primer used in the ObR ECDamplification, supra (i.e., 5′-AAGCTTTTCTGACCTGACNNN-3′ (SEQ ID NO:41))and that will enable the open reading frame in the ObR encodingnucleotide sequence to continue throughout the length of the IgGnucleotide sequence to be amplified. The template DNA in this reactionis the 2000 nucleotide segment of human IgG heavy chain genomic DNA(Ellison et al., 1982, Nuc. Acids. Res. 10:4071-4079).

The complete human obR-IgG fusion segment is prepared by an additionalPCR reaction. The purified products of the two PCR reactions above aremixed, denatured (95° C., 1 minute) and then renatured (54° C., 30seconds) to allow complementary ends of the two fragments to anneal. Thestrands are filled in using dNTPs and Taq polymerase and the entirefragment amplified using forward PCR primer of the first PCR reactionand the reverse PCR primer of the second PCR reaction. For convenienceof cloning into the expression vector, the resulting fragment is thencleaved with restriction enzymes which recognize unique designed sitesin the forward PCR primer of the first PCR reaction and the reverse PCRprimer of the second PCR reaction. This digested fragment is then clonedinto an expression vector that has also been treated with theserestriction enzymes.

Sequence analysis is used to confirm structure, and the construct isused to transfect COS cells to test transient expression.

Those skilled in the art are aware of various considerations whichinfluence the choice of expression vector into which the obR-IgG fusionsegment is to be cloned, such as the identity of the host organism andthe presence of elements necessary for achieving desired transcriptionaland translational control. For example, if transient expression isdesired, the obR-IgG fusion segment generated supra can be cloned intothe expression vector pcDNA-1 (Invitrogen). Alternatively, stableexpression of the fusion protein can be achieved by cloning the obR-IgGfusion segment into the expression vector pcDNA-3 (InVitrogen).

Alternatively, mouse and/or human obR-IgG fusion proteins can begenerated using an expression vector such as the CD5-IgG1 vector(described by Aruffo et al., 1990, Cell 61:1303-1313), which alreadycontains the IgG constant region. According to this method, the DNAfragment encoding the ObR extracellular domain is generated in a PCRreaction so that the open reading frame encoding the ObR extracellulardomain is continuous and in frame with that encoding the IgG constantregion.

For example, the extracellular domains (including signal peptides) ofmouse and human ObR were PCR amplified with Extaq (PanVera Corp.). Thefollowing primers were used for amplification of mouse and human ObR infirst generation expression constructs. Mouse: Forward primer,5′-CCCAATGTCGACATGATGTGTCAGAAATTCTAT-3′ (SEQ ID NO:45), Reverse primer,5′-AAAAAGGATCCGGTCATTCTGCTGCTTGTCGAT-3′ (SEQ ID NO:46). Human: Forwardprimer, 5′-CCCAATGTCGACATGGTGTACTTCTCTGAAGTA-3, (SEQ ID NO:47), Reverseprimer, 5′-TTTTTGGATCCCACCTGCATCACTCTGGTG-3′ (SEQ ID NO:48).

Each forward primer above contains a SalI restriction site and eachreverse primer above contains a BamHI restriction site. Afteramplification using the mouse and human obR cDNAs as templates, theresulting PCR fragments were cloned into the XhoI/BamHI sites of theCD5-IgG vector (Aruffo et al., 1990, Cell). The resulting vectors weretransiently transfected into COS cells and conditioned media wasgenerated. Immunoprecipitation (IP) of the conditioned media withprotein A and analysis by SDS PAGE revealed that the mouse ObR IgGfusion was expressed at greater levels than human ObR-IgG. To improveexpression of the human ObR-IgG fusion, primers were designed whichamplified the extracellular domain of human ObR (without the signalpeptide), and this fragment was coligated with sequences encoding thesignal-peptide of mouse ObR into the CD5-IgG vector. The followingprimers used for amplification of the human ObR ECD fragment that wasfused with mouse ObR signal peptide. Forward primer,

5′-TTTAACTTGTCATATCCAATTACTCCTTGGAGATTTAAGTTGTCTTGC-3′ (SEQ ID NO:49);reverse primer, 5′-TTTTTGGATCCCACCTGCATCACTCTGGTG-3′ (SEQ ID NO:50).

After amplification, restriction enzyme digestion, and subcloning, theresulting construct was transiently expressed in COS cells. IP andSDS-PAGE analysis of the resulting conditioned media showed successfulexpression of the 170 kDa human ObR IgG fusion. An alternative methodfor enhancing the expression of immunoglobulin fusion proteins, involvesinsertion of the ObR extracellular domain (not including the signalpeptide) into the CD5-IgG1 vector in such a manner that the CD5 signalpeptide is fused to the mature ObR extracellular domain. Such a signalpeptide fusion has been shown to improve expression of immunoglobulinfusion proteins.

10.3. Preparation of Modified Ch2 Domains

The nucleotide sequence of the obR-IgG gene fusion generated supra, canbe modified to replace cysteine residues in the hinge region with serineresidues and/or amino acids within the CH2 domain which are believed tobe required for IgG binding to Fc receptors and complement activation.

Modification of the CH2 domain to replace amino acids thought to beinvolved in binding to Fc receptor is accomplished as follows. Theplasmid construct generated supra, provides the template formodifications of the ObR-IgCγ1 CH2 domain. This template is PCRamplified using the forward PCR primer described in the first PCRreaction supra and a reverse primer designed such that it is homologousto the 5′ terminal portion of the CH2 domain of IgG1 except for fivenucleotide substitutions designed to change amino acids 234, 235, and237 (Canfield, S. M. and Morrison, S. L., 1991, J. Exp. Med.173:1483-1491) from Leu to Ala, Leu to Glu, and Gly to Ala,respectively. Amplification with these PCR primers yields a DNA fragmentconsisting of a modified portion of the CH2 domain. In a second PCRreaction, the template is PCR amplified with the reverse primer used inthe second PCR reaction supra, and a forward primer which is designedsuch that it is complementary to the Ig portion of the molecule andcontains the five complementary nucleotide changes necessary for the CH2amino acid replacements. PCR amplification with these primers yields afragment consisting of the modified portion of the CH2 domain, anintron, the CH3 domain, and 3′ additional sequences. The completeobR-IgCγ1 segment consisting of a modified CH2 domain is prepared by anadditional PCR reaction. The purified products of the two PCR reactionsabove are mixed, denatured (95° C., 1 minute) and then renatured (54°C., 30 seconds) to allow complementary ends of the two fragments toanneal. The strands are filled in using dNTP and Taq polymerase and theentire fragment is amplified using the forward PCR primer of the firstPCR reaction and the reverse PCR primer of the second PCR reaction. Forconvenience of cloning into the expression vector, the resultingfragment is then cleaved with restriction enzymes recognizing sitesspecific to the forward PCR primer of the first PCR reaction and thereverse PCR primer of the second PCR reaction. This digested fragment isthen cloned into an expression vector that has also been treated withthese restriction enzymes.

Sequence analysis is used to confirm structure, and the construct isused to transfect COS cells to test transient expression. hIgG ELISA isused to measure/confirm transient expression levels approximately equalto 100 ng protein/ml cell supernatant for the construct. CHO cell linesare transfected for permanent expression of the fusion proteins.

10.4. OBR-Ig Neutralizes Ob Protein

To establish whether the ObR-IgG fusion proteins were capable of bindingand neutralizing OB protein (leptin) in vitro and in mice, large scaletransient transfections were performed in 293 cells using the mouseObR-IgG fusion protein. The ObR-IgG protein was purified to nearhomogeneity on a protein A column and analyzed for its ability toinhibit the binding of an alkaline phosphatase-OB fusion protein (AP-OB)to cell surface ObR.

COS cells were transiently transfected with mouse obR cDNA and testedfor their ability to bind 0.5 nM AP-OB. As demonstrated in FIG. 10,purified ObR-IgG was able to potently inhibit, or neutralize, thebinding of AP-OB fusion protein to cell surface ObR.

FIG. 10, column 1, shows the high levels of specific binding observed inthe absence of ObR-IgG fusion protein. Columns 2, 3, and 4 show the nearcomplete inhibition of binding observed with three different columnfractions of purified ObR-IgG.

11. The OBR Long-Form has Signalling Capabilities of IL-6 Type CytokineReceptors

To address whether the cloned ObR isoforms are signaling competent, theObR gene was introduced into established tissue culture cell lines, andthe cell response to OB treatment was compared with that mediated by thestructurally-related IL-6 type cytokine receptors. The results presentedin this example provide evidence that the ObR long form is asignal-transducing molecule and shares functional specificity withIL-6-type cytokine receptors.

11.1. Materials and Methods 11.1.1 Cells

COS-1, COS-7, H-35 (Baumann et al., 1989, Ann. N.Y. Acad. Sci.557:280-297), HepG2, and Hep3B (Lai et al., 1995, J. Biol. Chem.270:23254-23257) cells were cultured as described. The cells weretreated in medium containing 0.5% fetal calf serum alone or supplementedwith 1 μM dexamethasone, 0.1-1000 ng/ml human OB, 1000 ng/ml mouse OB,IL-6 (Genetics Institute) or G-CSF (Immunex Corp.). To inhibit signalingby gp130, the cells were treated with the combination of twopan-blocking monoclonal antibodies against human gp130, B-R3 (Chevalieret al., 1995, N.Y. Acad. Sci. 762:482-484) and 144 (20 μg/ml).

11.1.2. Expression Vectors and Cat Reporter Gene Constructs

Expression vectors for the long form of human ObR and the short form ofmouse ObR are described above (Sections 7-9). The truncated humanG-CSFR(27) (Ziegler et al., 1993, Mol. Cell. Biol. 13:2384-2390) and ratSTAT1, STAT3 and STAT5B (Lai et al., 1995, J. Biol. Chem.270:23254-23257; Ripperger et al., 1995, J. Biol. Chem.,270:29998-30006) have been described. ObR with a mutated box 3 sequence(Y1141F) was generated by overlap extension PCR using syntheticoligonucleotides encoding the specified amino acid substitution (Higuchiet al., 1988, Nucleic Acids Res. 12:5707-5717). The y1141F contains areplacement of the tyrosine at position 1141 with phenyalanine. PlasmidSV-SPORT1 (Life Technologies, Inc.) containing rat STAT3 truncated by 55carboxy-terminal residues has been generated by converting codons 716and 717 into two stop codons. The CAT reporter gene constructs,pHRRE-CAT and pIL-6RE-CAT, have been described previously (Lia et al.,1995, J. Biol. Chem. 270:23254-23257; Morella et al., 1995, J. Biol.Chem. 270:8298-8310).

11.1.3. Cell Transfection and Analysis

COS-1, H-35 and Hep3B cells were transfected with plasmid DNA by theDEAE-dextran method (Lopata et al., 1989, Nucleic Acids Res.12:5707-5717); HepG2 cells by the calcium phosphate method (Graham etal., 1973, Virology 52:456-461); and COS-7 cells by the lipofectaminemethod. Subcultures of COS cells were maintained for 16 hours inserum-free medium prior to the activation of STAT proteins by treatmentwith cytokines for 15 minutes. DNA binding by STAT proteins wasdetermined by EMSA on whole cell extracts as described in Sadowski etal. (1993, Science 26:1739-1744). Double stranded oligonucleotides forthe high affinity SIEm67 (Sadowski et al., 1993, Science 26:1739-1744)and TB-2 (Ripperger et al., 1995, J. Biol. Chem. 270:29998-30006) servedas EMSA substrates. CAT gene-transfected cell cultures were treated for24 hours with cytokines or OB. CAT activities were quantitated bytesting serial dilutions of cell extracts, normalized to the expressionof the cotransfected marker plasmid pIE-MUP (Morella et al., 1995, J.Biol. Chem. 270:8298-8310), and are expressed relative to the value ofthe untreated control cultures in each experimental series (defined as=1.0). Quantitative cell surface binding of the AP-OB fusion protein(Section 6) was done essentially as outlined by Cheng and Flanagan(1994, Cell 79:157-168).

11.2. Results and Discussion 11.2.1. OBR Activates Stat Proteins

To determine whether ObR has the ability to recruit the cellularsignaling machinery, COS cells were transiently transfected withexpression vectors for the two representative forms of ObR, mouse shortform (also corresponding to a mutated form detected in db/db mice) andhuman long form. Two days after transfection, cells were incubated in 1nM human or mouse alkaline phosphatase-OB, and cell surface expressionof ObR was detected as indicated by specific binding of the alkalinephosphatase-OB (AP-OB) fusion protein. Transfection of the short formObR resulted in approximately 10-fold higher binding than the long form.Scatchard transformation of binding data performed at multiple AP-OBconcentrations indicated that the lower binding observed for the longform was mainly a result of reduced cell surface expression. The mouseshort form bound both the murine and human ligands with an affinity of0.7 nM, and the human long form bound both the murine and human ligandswith an affinity of 1.0 nM.

COS-1 cells were co-transfected with expression vectors for human ormouse ObR (2 μg/ml) and the various STAT proteins (3 μg/ml).Co-transfection of the expression vectors for ObR and various STATisoforms allowed analysis of the ligand-induced activation of specificSTAT proteins. The transfected cells were treated for 15 minutes withoutor with murine OB (100 ng/ml) and activation of DNA binding of the STATproteins was identified by EMSA using the diagnostic oligonucleotidesubstrates STE or TB-2. In these experiments, only the long form of ObRactivated either endogenous COS STAT proteins, or the co-expressedSTAT1, STAT3, or STAT5B. Activation of all STAT isoforms by ObR wasligand dependent. In contrast, the short form of ObR was unable toactivate any endogenous or co-transfected STAT proteins despite its highsurface expression. Since the long form of ObR activated all the STATproteins that are also activated by G-CSFR, LIFR, and gp130 (Kishimotoet al., 1995, Blood 86:1243-1254; Lia et al., 1995, J. Biol. Chem.270:23254-23257), the long form ObR was predicted to stimulatetranscription with a specificity of the IL-6-type cytokine receptors.

11.2.2. OBR Signals Induce Gene Expression

Rodent and human hepatoma cell lines have previously been utilized todefine the gene-inducing action of ectopically-expressed hematopoietinreceptors (Baumann et al., Mol. Cell. Biol. 14:138-146). Consequently,three complementary hepatoma cell lines were applied to characterize ObRsignaling. The long or short forms of ObR or human G-CSFR, wereintroduced into rat H-35 cells, together with the HRRE-CAT reporter geneconstruct, the expression of which is increased in these cells bysignals of many hematopoietin receptors (Morella et al., 1995, J. Biol.Chem. 270:8298-8310). Subcultures were treated for 24 hours withserum-free medium alone or containing cytokines (mOB, LIF, or IL-6) withor without dexamethasone. The long form of ObR mediated ligand-dependentinduction of CAT gene expression. The stimulatory action wassynergistically enhanced by dexamethasone. The cell response mediated byObR was highly similar to that of the endogenous IL-6R butcharacteristically different from the endogenous LIFR. In contrast, theshort form of ObR failed to induce gene expression, indicating that the34 residue cytoplasmic domain, despite the presence of a box 1-relatedmotif, was ineffective in recruitment of the cellular signalingcomponents. The fact that the G-CSFR with a cytoplasmic domain truncatedto 27 residues still induced gene transcription in the presence ofligand illustrated that the cells were able to respond to the signalderived from a short, box-1-containing cytoplasmic domain of ahematopoietin receptor. The lack of induction of CAT gene expression inG-CSFR-transfected control cells demonstrates that H-35 cells do notrespond to OB in the absence of transfected ObR.

11.2.3. OBR Functions Independently of gp130

The results described above support the model that the long form of ObRreconstitutes a signaling pathway similar to that of IL-6R. Next, todetermine whether gp130 is part of the functional ObR, the long form ofObR was introduced together with HRRE-CAT or IL-6RE-CAT into HepG2 cellsand the inhibitory effects of anti gp130 antibodies was assessed.

Treatment of the transfected HepG2 cells with either mouse or human OBproduced a similarly strong induction which was in the range of thatproduced by IL-6 (30-40 fold stimulation). A dose response analysisindicated that maximal regulation was achieved with 100 ng/ml OB. Infour independent experiments, it was established that 1-5 ng/ml OBproduced a half-maximal stimulation, and that 1000 ng/ml yielded astimulation that was consistently below maximum. In the presence ofmonoclonal antibodies against human gp130, which are known to preventsignaling by all IL-6 type cytokine receptors (Chevalier et al., 1995,N.Y. Acad. Sci. 762:482-484), the stimulation of gene expression by IL-6was abolished as expected, whereas the regulation by OB was unaffected.These results indicate that ObR functions independently of gp130(insensitive to anti-gp130) and that signal initiation may be triggeredby receptor homo-oligomerization.

11.2.4. Box 3 Sequence of OBR AND STAT3 are Involved 1N Signaling

Induction of transcription via IL-6 RE is characteristic of thehematopoietin receptors of IL-10R which contain at least one copy of thebox 3 motif (YXXQ) in their cytoplasmic domains (Lai et al., 1995, J.Biol. Chem. 270:23254-23257). This box 3 sequence has been implicated inrecruiting STAT3 to the receptor as part of its activation byreceptor-associated kinases (Lia et al., 1995, J. Biol. Chem.270:23254-23257); Stahl et al., 1995, Science 267:1349-1353). The longfonn of ObR (FIGS. 3A-3F) contains at amino acid position 1141 to 1144one copy of the box 3 motif that could account for the activation ofSTAT3 and transcriptional stimulation of IL-6RE-CAT. To assess whetherthe box 3 motif of ObR and STAT3 were involved in the gene inducingeffect of QbR, two complementary reagents were applied: a box 3-mutantObR and a dominant negative STAT3. The role of box 3-sequence in thelong form of ObR was determined by mutating tyrosine at amino acidposition 1141 to phenylalanine (Y1141F). Hep G2 and H-35 cells weretransfected with an expression vector for wild-type ObR or ObRY1141F (2μg/ml) together with either pHRRE-CAT or pIL-6RE-CAT. Cells were treatedwith human OB (100 ng/ml), and the relative change in CAT activity wasdetenninecE The mutant ObR transfected into HepG2 cells yielded a lowerstimulation of both the HRRE-and IL-6RB-CAT reporter gene constructsthan the wild-type type ObR. For example, stimulation of HRRE-CATexpression was reduced 40 fold in HepG2 cells and H-35 cells.Stimulation of IL-6RE-CAT was reduced 20-fold in HepG2 cells and100-fold in H-35 cells. Control experiments indicated that reducedsignaling activity of the mutant ObR was not due to compromised surfaceexpression as shown by AP-OB binding. The relative effect of themutation was more prominent on IL-6RE than on HRRE. A similar experimentcarried out in H-35 cells showed that box 3 mutation was correlated witha loss of IL-6RE regulation, whereas HRRE regulation was minimallyaffected. The results are consistent with previous observations that, insome cell lines, the recruitment of STAT3 was more important in geneinduction through IL-6RE then through HRRE (Lal et al., 1995, J. BiolChem. 270:23254-23257; Morella et al., 1995, J. Biol Chem.270:8298-8310; Wang et al., 1995 Blood 86:1671-1679).

The reduced gene-regulatory effect of the Y1141F ObR mutant was alsocorrelated with a lower activation of STAT proteins. When the mutant ObRwas transfected into COS-1 cells, as done for the wild-type ObR,activation of the endogenous COS STAT proteins was not detected. Also,ObR Y1141F was approximately 10 times less effective in activatingoverexpressed STAT1 and STAT3 than wild type ObR. Activation of STAT5Bwas, however, unaffected by the mutation. This profile of STATactivation by ObR Y1141F was in agreement with that observed for box3-deficient gp130 (Lai et al., 1995, J. Biol. Chem. 270:23254-23257) andG-CSFR (Morella et al., 1995, J. Biol. Chem. 270:8298-8310) and wouldexplain the specific changes in the regulation of the reporter geneconstructs.

The signal transducing role of STAT3 was determined by usingover-expression of STAT3_(—)55C, a mutant STAT3 with a 55 residuecarboxy terminal truncation that acts as dominant negative inhibitor ofSTAT3 action on gene transcription. DNA binding assays such as thosedescribed in Section 11.2.1., supra, verified that the long form of ObRefficiently activated DNA binding activity of STAT3_(—)55C. STAT3_(—)55Cessentially abolished the ObR mediated induction of IL-6RE and reducedthat of HRRE by 50%. These data indicate that in the hepatic cells, ObRengages signal transduction pathways that are also utilized by theIL-6-type cytokine receptors and are sensitive to STAT3_(—)55C.

11.2.5. OBR Can Utilize Both STAT3 and STAT5B Gene Induction

Induction of the selected reporter gene constructs in HepG2 or H-35cells is maximal and not significantly enhanced by over-expressedwild-type STAT proteins. To assess whether the STAT proteins activatedby ObR play a positive mediator role, human Hep3B cells were transfectedwith human ObR together with either pIL-6RE-CAT or pHRRE-CAT, and theexpression vector for the STAT proteins. Stimulation of CAT activity byhuman OB (100 ng/ml) relative to untreated control was determined(mean±S.D.; N=3 to 4). Those hepatoma cells have retained expression offunctional IL-6R, but lack the receptors to other IL-6-type cytokines(Baumann et al., 1994, Mol. Cell. Biol. 14:138-146). Moreover, thesecells have a relatively low level of STAT3 and STAT5, thus permittingtesting of the signaling of ObR by gain of function throughover-expression of STAT proteins. The results from these experimentsindicate that overexpressed STAT3 mediated induction of IL-6RE 15-fold.Overexpressed STAT31 and STAT5B enhanced ObR mediated induction ofHRRE-CAT 5-fold and 30-fold, respectively.

11.3. Conclusion

The results presented above document that full length ObR is a signaltransducing receptor with a mode of action related to the IL-6-typecytokine receptors. The data also support the hypothesis that thetruncated ObR variants, such as the short form expressed in many tissuesor encoded by the db mutant transcript, are either signaling-incompetentor exert a reduced signaling repertoire that is not detectable by thetools applied here. The fact that reconstitution of an OB response isachieved at the level of gene expression in hepatic cells stronglysuggests that an equivalent process may occur in hypothalamic cells orother cell types that normally express the full-length ObR. The link ofObR to specific signaling pathways utilizing STAT proteins and theknowledge of the specificity of these proteins to control genes throughidentifiable DNA binding elements may assist in identifying theimmediate ObR effects that are relevant to understanding OB action invivo. The experimental system presented above can also be used toaddress questions about the functional role, if any, of the naturallyoccurring short forms of ObR in functional regulation of the long form.

12. Mutational Analysis of OBR

In order to identify regions of the ObR cytoplasmic region important foractivation of genes, a number of ObR mutants were created and analyzed.These studies, described below, identified two distinct regions of theObR cytoplasmic domain important for induction of gene expression.

12.1 Materials and Methods 12.1.1 Cells

COS-1, COS-7, and H-35 cells were cultured as described by Baumann etal., 1989, Ann. N.Y. Acad. Sci. 557:280-297. Cells were mock stimulatedin medium containing 0.5% fetal calf serum and 1 μM dexamethasone ortreated in the same medium supplemented with 100 ng/ml human leptin(Roche), IL-6 (Genetics Institute), or G-CSF (Immunex Corp.).

12.1.2 Expression Vectors and Cat Reporter Gene Constructs

The expression vectors for the long form of human ObR are describedabove (Section 9) and rat STAT1, STAT3 and STAT5B have been describedpreviously (Lai et al., 1995, J. Biol. Chem. 270:23254-23257; Rippergeret al., 1995, J. Biol. Chem. 270:29998-30006). pOB-R_(—)1115-1165,pOB-R_(—)1065-1165 and pOB-R_(—)965-1165, all encoding carboxy-terminaltruncated human ObRs, were generated by PCR. Briefly, oligonucleotidesspanning the intracellular domain of human ObR were used to generatein-frame stop codons 3′ to the specified amino acids. The PCR fragmentswere digested with EcoRV and XbaI and subcloned into human ObR that hadbeen digested with EcoRV and XbaI. A similar strategy was used togenerate pOB-R_(—)868 but with primers generating an MscI-XbaI fragmentthat replaced endogenous human ObR sequences. pOB-RY1141F, encodinghuman ObR with a mutated box 3 sequence was prepared as described inSection 11.1.2. ObR mutants pOB-R(box1mt), containing PNP to SNS changesin the ObR box 1 motif (aa 876 and 878), and mutants pOB-RY986F andpOB-RY1079F, were generated by overlap extension PCR using syntheticoligonucleotides encoding the specified Tyr to Phe amino acidsubstitutions (Higuchi et al., 1988, Nucleic Acids Res. 16:7351-7367).The CAT reporter gene constructs, pHRRE-CAT and pIL-6-CAT have beendescribed previously (Lai et al., 1995, J. Biol. Chem. 270:23254-23257;Morella et al., 1995, J. Biol. Chem. 270:8298-8310).

12.1.3 Cell Transfection and Analysis

COS-1 and H-35 cells were transfected by the DEAE-dextran method (Lopataet al., 1984, Nucleic Acids Res. 12:5707-5717), and COS-7 cells by thelipofectamine method (Tartaglia et al., 1995, Cell 83:1263-1271). Foranalysis of STAT protein activation, COS cells were maintained for 16hours in serum-free medium, followed by treatment of cells with 100ng/ml leptin or G-CSF for 15 minutes.

For CAT assays, transfected cell cultures were subdivided and treatedwith ligands for 24 hours. CAT reporter activities were determined andare expressed relative to values obtained for untreated control culturesfor each experimental series. DNA binding by STAT proteins was analyzedby electromobility shift assay (EMSA) using whole cell extracts asdescribed by Sadowski et al. (1993, Science 26:1739-1744). Radiolabeleddouble stranded oligonucleotides SIEm67 (for STAT1 and STAT3) and TB-2(for STAT5B) served as binding substrates in the EMSA. Receptorexpression in COS cells was analyzed by quantitative cell surfacebinding of AP-OB fusion protein as described by Cheng and Flanagan(1994, Cell 79:157-168).

12.1.4 Immunoblotting

All immunoblotting was performed as described by Baumann et al. (1996,Proc. Natl. Acad. Sci. U.S.A. 93:xxx-xxx) and immunoreactive proteinswere visualized by enhanced chemiluminescence detection as described bythe manufacturer (Amersham). Rabbit polyclonal antiserum specific forSTAT5B was obtained from Santa Cruz Biotechnology.

12.2 Results and Discussion

As discussed above, ObR is a member of the class I cytokine receptorsuperfamily. Receptors of this class lack intrinsic tyrosine kinaseactivity and are activated by ligand-induced receptor homo-dimerizationor hetero-dimerization. In many cases, activation requires activation ofreceptor-associated kinases of the Janus family (JAKs) (Ihle et al.,1994, Trends. Biol. Sci. 19:222-227). JAKs associate with themembrane-proximal domain of the intracellular part of the cytokinereceptors, and serve to initiate signal transduction pathways followingligand induced receptor activation. Included among the downstreamtargets of the JAK proteins are members of the STAT (Signal Transducersand Activators of Transcription) family of transcription factors (Ihleet al., 1994, Trends. Biol. Sci. 19:222-227). The STATs are DNA bindingtranscription factors that contain Src-homology (SH2) domains thatinteract with receptor molecules through phosphorylated tyrosineresidues. STAT proteins are activated by tyrosine phosphorylation, formheterodimers or homodimers, translocate to the nucleus, and modulatetranscription of target genes.

12.2.1 The OBR Intracellular Domain Includes at Least Two RegionsImportant for Signalling

To define regions of the ObR cytoplasmic domain required for signaling,a series of C-terminal deletion mutants were constructed (FIG. 11A).cDNAs encoding these mutants were transiently co-transfected into H-35cells with either IL-6RE-CAT or HRRE-CAT reporter constructs and assayedfor their ability to stimulate transcription (FIG. 11B). C-terminaltruncations that remove box 3 sequences (aa 1141-1144) of ObR abolishtranscriptional activation via IL-6-RE (FIG. 11B; upper panel). Thisresult is consistent with the fact that a Y to F mutation in the singlebox 3 motif or ObR completely disrupts signaling in H-35 cells viaIL-6RE (Section 11.2.4). In contrast, ObR signaling through HRRE wasminimally reduced by removal of extreme C-terminal sequences and was notcompletely disrupted until removal of the approximately 97 amino acidsbetween 868 and 965 (FIG. 11B).

To ensure that the expression vectors for the various ObR mutantsdirected the synthesis of surface localized receptor proteins, COS cellstransfected with each construct were assayed for receptor expression byAP-OB binding studies. C-terminal truncations of ObR generate proteinsthat are expressed at the surface and bind ligand (FIG. 12). Moreover,the expression level of ObR increased with progressive truncation of theintracellular domain.

As discussed above, ObR gene induction via IL-6RE correlates withactivation of STAT1 and STAT3 whereas ObR gene induction via HRRE wasfound to correlate with activation of STAT5B. To further evaluate thecorrelation between HRRE stimulation and STAT5B activation, COS cellswere co-transfected with expression plasmids for STAT5B and the ObRdeletion mutants. Immunoblotting performed on extracts prepared fromthese cells revealed that STAT5B was expressed at relatively equalamounts in each of the transfected cultures. Cells were treated withleptin. EMSA analysis was performed, and STAT protein levels werequantitated by Western blotting. Progressive C-terminal truncations ofObR result in a reduced ability to activate STAT5B and detectable STAT5Bactivation was lost only with removal of the membrane proximal ObRsegment (construct pOBR_(—)868-1165). Thus, there appears to be acorrelation between loss of ObR STAT5B activation and gene induction viaHRRE.

To define the relative contribution of the conserved intracellulardomain tyrosine residues and of the membrane proximal box 1 motif tosignaling by ObR via HRRE, mutants OB-RY1141F, OB-RY986F, OB-RY1079F andOB-R(box 1mt) were generated (FIG. 13A). When analyzed in COS cells,AP-OB binding studies demonstrate that these mutants are expressed atthe cell surface approximately as well as wild-type ObR. Whentransfected into H-35 cells, OB-RY986F and OB-RY1079F were unchanged intheir ability to regulate HRRE (FIG. 13B). In contrast, mutation of theObR box 1 motif results in a complete loss of regulation of geneinduction through this element. Thus, the box 1 motif of ObR appears tobe an important determining factor for the ability of ObR to activatepathways that can modulate gene induction via HRRE.

Gene induction by ObR through IL-6RE requires sequences near the extremeC-terminus of ObR (FIG. 11B). In contrast, ObR gene induction throughHRRE does not appear to require these C-terminal sequences. Moreover,gene induction via this element is only minimally affected by removal ofObR intracellular domain sequences of approximately 200 amino acidsbetween amino acids 965-1165 but is dependent upon membrane proximalsequences of the approximately 17 amino acids between amino acids 868and 965. Consequently, the proposed box 2 motif of ObR (Lee et al.,1996, Nature 379:632-635) (human ObR aa 1066-1075) does not appear tocontribute to gene induction through HRRE. EMSA analysis suggests geneinduction of HRRE correlates with the ability of ObR to activate STAT5B.Interestingly, OB-R_(—)965-1165, which has been deleted of allintracellular domain tyrosine residues and therefore all potential SH2docking sites, is still capable of low-level STAT5B activation andtranscriptional stimulation through HRRE. Only when membrane proximalsequences of ObR are removed (OB-R_(—)868-1165), are both HRRE geneinduction and STAT5B activation completely abolished. Consistent withthis, OB-R (box-1mt), containing a mutated box 1 motif, is similarlyunable to induce gene induction through HRRE and would be predicted tobe unable to activate STAT5B.

13. Multimerization of OBR

The primary structure of ObR suggests that it is closely related to thesignaling subunits of the IL-6-type cytokine receptors. Members of thisgroup can be activated by either heterodimerization or homodimerization(Kishimoto et al., 1994, Cell 76:253-262; Heldin et al., 1995, Cell80:213-223). Included among the former are the receptors for IL-6,leukemia inhibitory factor (LIF), oncostatin M, IL-11, and ciliaryneurotrophic factor (CNTF), all of which share the common signaltransducer, gp130 (Kishimoto et al., 1994, Cell 76:253-262; Taga et al.,1989, Cell 58:573-581). However, ObR appears to signal independently ofgp130 (Baumann et al., 1996, Proc. Natl. Acad. Sci. USA 93:xxx-xxx.Therefore, ObR may function in the presence of another accessory chainsuch as the common signaling subunit utilized by receptors for eitherIL-3, granulocyte macrophage-colony stimulating factor (GM-CSF) and IL5(IL-3Rβ), or IL-2, IL-4, IL-7 and IL-9 (IL-2Rγ). However, ObR signals inhepatoma cells, which do not express either IL-3Rβ or IL-Rγ (Wang etal., 1995, Blood 86:1671-1679; Morella et al., 1995, J. Biol. Chem.270:8298-8310). Alternatively, ObR may be activated by homodimerizationas is found for the granulocyte-colony stimulating factor receptor(G-CSFR) (Fukanaga et al., 1991, EMBO J. 10:2855-2865; Ishezaka-Ikeda etal., 1993, Proc. Natl. Acad. Sci. USA 90:123-127). Therefore, todetermine whether ObR has the ability to dimerize and signal as ahomodimer, chimeric receptors encoding the extracellular domain ofG-CSFR joined to the intracellular domain of ObR or the reciprocalreceptor having the extracellular domain of ObR joined to theintracellular domain of G-CSFR were constructed and analyzed (FIG. 14A).

13.1 Materials and Methods 13.1.1 Cells

COS-1, COS-7, and H-35 cells were cultured as described by Baumann etal., 1989, Ann. N.Y. Acad. Sci. 557:280-297. Cells were mock stimulatedin medium containing 0.5% fetal calf serum and 1 μM dexamethasone ortreated in the same medium supplemented with 100 ng/ml human leptin(Roche), IL-6 (Genetics Institute), or G-CSF (Immunex Corp.).

13.1.2 Expression Vectors and Cat Reporter Gene Constructs

The expression vectors for the long form of human ObR are describedabove (Section 9), full-length G-CSFR or truncated G-CSFR(_cyto)(Ziegler et al., 1993, Mol. Cell. Biol. 13:2384-2390), and rat STAT1,STAT3 and STAT5B have been described previously (Lai et al., 1995, J.Biol. Chem. 270:23254-23257; Ripperger et al., 1995, J. Biol. Chem.270:29998-30006). As used herein the term “_cyto” means deletion of thecytoplasmic domain. The G-CSFR/ObR chimeric receptor was generated byPCR and encodes the extracellular domain of human G-CSFR (aa 1-598)joined near the transmembrane and intracellular domain of human ObR (aa829-1165). The ObR/G-CSFR chimeric receptor was generated by PCR andencodes the mouse ObR extracellular domain and transmembrane sequences(aa 1-860) joined to the intracellular domain of the human G-CSFR (aa631-813). The CAT reporter gene constructs, pHRRE-CAT and pIL-6-CAT havebeen described previously (Lai et al., 1995, J. Biol. Chem.270:23254-23257; Morella et al., 1995, J. Biol. Chem. 270:8298-8310).

13.1.3 Cell Transfection and Analysis

COS-1 and H-35 cells were transfected by the DEAE-dextran method (Lopataet al., 1984, Nucleic Acids Res. 12:5707-5717), and COS-7 cells weretransfected by the lipofectamine method (Tartaglia et al., 1984, Cell83:1263-1271). For analysis of STAT protein activation, COS cells weremaintained for 16 hours in serum-free medium, followed by treatment ofcells with 100 ng/ml leptin or G-CSF for 15 minutes.

For CAT assays, transfected cell cultures were subdivided and treatedwith ligands for 24 hours. CAT reporter activities were determined andare expressed relative to values obtained for untreated control culturesfor each experimental series. DNA binding by STAT proteins was analyzedby electromobility shift assay (EMSA) using whole cell extracts asdescribed by Sadowski et al. (1993, Science 26:1739-1744). Radiolabeleddouble stranded oligonucleotides SIEm67 (for STAT1 and STAT3) and TB-2(for STAT5B) served as binding substrates in the EMSA. Receptorexpression in COS cells was analyzed by quantitative cell surfacebinding of AP-OB fusion protein as described by Cheng and Flanagan(1994, Cell 79:157-168).

13.1.4 Immunoblotting

All immunoblotting was performed as described by Baumann et al. (1996,Proc. Natl. Acad. Sci. USA 93:xxx-xxx) and immunoreactive proteins werevisualized by enhanced chemiluminescence detection as described by themanufacturer (Amersham). Rabbit polyclonal antiserum specific for STAT5Bwas obtained from Santa Cruz Biotechnology. Goat polyclonal antiserumagainst bacterially expressed extracellular domain of G-CSFR wasprepared at Roswell Park Cancer Institute Springville Laboratories.

13.2 Results and Discussion

The experiments described below suggest that, while dimerization of theObR cytoplasmic domain may be sufficient for signal transduction, higherorder homo-oligomers can be formed in response to ligand binding.

13.2.1 Homodimerization Obr Intracellular Domains May be Sufficient forSignal Transduction

Since chimeric receptor complexes have proven quite productive for theanalysis of the mechanism of cytokine receptor activation (Morella etal., 1995, J. Biol. Chem. 270:8298-8310; Vigon et al., 1993, Oncogene8:2607-2615; Baumann et al., 1994, Mol. Cell. Biol. 14:138-146),ObR/G-CSFR and G-CSFR/ObR chimeras were produced and studied as a meansto analyze the mechanism of ObR signaling (FIG. 14A). To analyze whetherthe G-CSFR/ObR chimeric receptor could propagate a ligand induced signalcomparable to that for wild-type ObR, the chimera was tested for STATactivation and for transcriptional stimulation. Co-transfection ofG-CSFR/ObR with STAT proteins yielded a G-CSF-induced activation ofSTAT1, STAT3, and STAT5B. This result is similar to the STAT proteinactivation induced by OB in ObR transfected cells (Section 12).Expression of the chimeric receptor was confirmed by immunoblot analysisof cultures transfected with G-CSFR/ObR. These results suggest thatG-CSF mediated dimerization of ObR cytoplasmic domains can generate anObR-type activation of STAT proteins. In addition, it was found that theG-CSFR/ObR chimera could stimulate transcription as detected bymeasurement of gene induction in H-35 cells following receptorco-transfection with the IL-6RE and HRRE reporter constructs (FIG. 14B).The response elicited was found to be similar to an induction of thereporter gene constructs by either ObR or endogenous IL-6R.

These results indicate that homodimerization of two ObR cytoplasmicdomains can initiate signaling by ObR, similar to the mechanismmediating signaling by wild-type G-CSFR. However, the G-CSFR/ObR chimeracould not definitively prove that OB ligand has the capability todimerize ObR extracellular domains. Consequently, signaling activity bythe reciprocal chimera, containing the ObR extracellular domain joinedto the G-CSFR intracellular domain, was analyzed (FIG. 14A). Indeed, theObR/G-CSFR chimera could mediate gene induction comparable to that bywild-type ObR, G-CSFR/ObR, and wild-type G-CSFR (FIG. 14B). Thus, takentogether, these results suggest that ObR does not require an accessorychain for signaling, and that aggregation of two ObR intracellulardomains appears sufficient for receptor activation.

The fact that aggregation of two ObR intracellular domains is sufficientto generate a signal following ligand-induced activation suggests thatObR may function by receptor homodimerization. If so, signaling by ObRmight be “poisoned” by overexpression of a homodimerizing partner thatis signaling deficient, similar to what has been shown for members ofthe receptor tyrosine kinase family (Paulson et al., 1989, J. Biol.Chem. 264:17615-17618; Svensson et al., 1990, J. Biol. Chem.265:20863-20868; Wen et al., 1992, J. Biol. Chem. 267:2512-2518; Fantlet al., 1993, Annu. Rev. Biochem. 62:453-481). As discussed above(Section 12), ObR containing only the membrane proximal 6 amino acids ofthe cytoplasmic domain is signaling defective (FIG. 11B). Consequently,experiments were performed to determine whether expression of atruncated, signaling deficient ObR could disrupt signaling byfull-length ObR. Cells were co-transfected with increasing amounts oftruncated receptor OB-R_(—)868-1165 relative to full-length ObR and theability of these complexes to stimulate expression of a reporter geneconstruct was assayed. Co-transfection of increasing amounts oftruncated ObR does result in decreased signaling by wild-type receptor(FIG. 15A). However, even when there was a large excess of truncatedreceptor, relative to full-length receptor, the signaling repressionobserved did not approach the degree of reduction observed forrepression of G-CSFR signaling by overexpressed and signaling-deficienttruncated G-CSFR(_cyto) (FIG. 15A and FIG. 15C). The differingsensitivity to dominant negative repression observed for ObR and G-CSFRwas a property of their extracellular domains as shown by dominantnegative studies with the receptor chimeras (FIG. 15B and FIG. 15C).

A potential explanation for this weak dominant negative repression ofObR is that interaction of two ObR molecules may require functionaldomains residing in the intracellular region of the receptor. To addressthis possibility, the dominant negative repression of ObR by a mutantreceptor rendered signaling defective by a single amino acidsubstitution (Y1141F) in the ObR box 3 motif was examined. As describedabove, this mutation completely abolished the ability of ObR to modulategene induction via IL-6RE in H-35 cells (Section 12). Consequently, theability of OB-R(Y1141F) to inhibit wild-type ObR signaling via thisenhancer element was investigated. These studies revealed thatincreasing the ratio of transfected mutant OB-RY1141F to wild-typereceptor did not strongly repress signaling (FIG. 15E). Thus, the ObRbox 3 mutant and OB-R_(—)868-1165 behave similarly in their ability totrans-repress signaling by wild-type ObR. Interestingly, low levelexpression of either truncated or box 3 mutant ObR receptor generates aslight enhancement of signaling by wild-type ObR. Moreover, a similarpattern was also observed for ObR/G-CSFR signaling in the presence ofincreasing amounts of truncated OB-R_(—)868-1165 (FIGS. 15A, 15B, and15C).

As discussed above (Section 11), ObR can signal in hepatoma cells in thepresence of neutralizing antibodies to the gp130 signal transducingcomponent of the IL-6-type cytokine receptors. Moreover, these hepatomacells do not express the other characterized cytokine receptor accessorychains IL-2Rγ or IL-3Rβ (Wang et al., 1995, Blood 86:1671-1679; Morellaet al., 1995, J. Biol. Chem. 270:8298-8310). Consequently, it ispossible that ObR may function by a mechanism involving receptorhomodimerization. Among members of the class I cytokine receptor family,signaling by the G-CSFR is predicted to be initiated by ligand-inducedreceptor homodimerization (Fukanaga et al., 1991, EMBO J. 10:2855-2865;Ishezaka-Ikeda et al., 1993, Proc. Natl. Acad. Sci. USA 90:123-127). Asstated above, chimeric receptor complexes have proven quite productivefor the analysis of the mechanism of cytokine receptor activation(Morella et al., 1995, supra; Vigon et al., 1993, supra; Baumann et al.,1994, supra), ObR/G-CSFR and G-CSFR/ObR chimeras were produced andstudied as a means to analyze the mechanism of ObR signaling. Thesestudies revealed that the G-CSFR/ObR chimera can strongly activatetranscription of both the IL-6RE-CAT and HRRE-CAT reporter constructs(FIG. 14B). Since G-CSFR is thought to form a homodimer when G-CSF isbound, this implies that the aggregation of two intracellular ObRdomains is sufficient to initiate receptor signaling. In a similarmanner, the ObR/G-CSFR chimera also mediates transcriptional activationthrough IL-6RE and HRRE (FIG. 14B). These results show that leptinbinding can dimerize two ObR extracellular chains thus inducing theassociation of at least two intracellular G-CSFR domains and activationof the receptor complex. Moreover, these results suggest that it may bepossible to generate small molecules, peptides, or antibodies that actas ObR agonists through simple crosslinking of two ObR chains.

As would be predicted for receptors that are activated by simplehomodimerization, signaling by full length G-CSFR and the G-CSFR/ObRchimera can be greatly diminished by co-expression of a signalingdeficient homodimerizing partner. However, OB-R_(—)868-1165 was unableto as efficiently repress signaling by full-length ObR or the ObR/G-CSFRchimera. It is therefore possible that leptin binding to cell surfacereceptors can result in higher-order oligomerization (receptornumber>2/complex) as has been shown for IL-10 receptor complexes (Tan etal., 1995, J. Biol. Chem. 21:12906-12911) and for members of theActivin/TGF-βR family (Brand et al., 1993, J. Biol. Chem.268:11500-11503; Weiser et al., 1993, Mol. Cell. Biol. 13:7239-7247;Wrana et al., 1994, Cell 71:1003-1014; Moustakas et al., 1993, J. Biol.Chem. 268:22215-22218; Henis et al., 1994, J. Cell Biol. 126:139-154).According to this model, ligand binding by full-length ObR or ObR/G-CSFRchimera can lead to aggregation of more than two receptor chains, yetjuxtaposition of only two intracellular domains is sufficient for signalgeneration. Such complexes would be predicted to be highly resistant todominant negative repression. The strong repression of signaling byG-CSFR(_cyto) in complexes containing the G-CSFR/ObR chimerademonstrates that ObR intracellular domain can be efficiently repressedwhen placed in the context of a simple homodimer structure. Although itis possible that OB-R_(—)868-1165 localizes to a different region of themembrane than wild-type ObR, it is not likely that mutation of a singletyrosine residue of the ObR intracellular domain (Y1114F) would resultin altered receptor membrane localization. Thus, the observation ofsimilar repression effects mediated by either OB-R_(—)868-1165 orOB-RY1141F suggests the results described herein are not due to alteredmembrane localization. Low expression levels of either OB-R_(—)868-1165and OB-RY1141F generate a small enhancement of signaling for full lengthObR and the ObR/G-CSFR chimera. This effect could be attributable toeither ligand presentation (Andres et al., 1989, J. Cell Biol.109:3137-3145; Massaugue et al., 1992, Cell 69:1067-1070; Lin et al.,1993, Trends. Cell Biol. 3:14-19), or ligand passing as has previouslybeen observed for the TNF receptor (Tartaglia et al., 1993, J. Biol.Chem. 268:18542-18548).

As noted above, it is possible that the short forms of ObR serve atransport or clearance function in the body (Tartaglia et al., 1995,Cell 83:1263-1271). However, the possiblity that the long and shortforms of ObR can functionally interact suggests that the short form ofObR could regulate activities of the long form. This is supported by thefact that the major naturally occurring non-signalling short form of ObRin the mouse (containing a 34 amino acid intracellular domain), whichalso corresponds to the mutant ObR found in the db/db mouse, can represslong form receptor signaling.

14. A Cell-Based Assay for Identification of Agonists And Antagonists ofthe Ob Receptor Signalling Pathway

The following example describes methods for identifying compounds thatcan be used to treat a body weight disorder. This assay works byidentifying compounds (e.g., small molecules, peptides, or antibodies)that function as either agonists or antagonists of the Ob receptor or ofany other component of the pathway (e.g., a component of the Ob receptorsignalling pathway described above) that is influenced by receptorbinding. Compounds that are discovered by performing the assay describedherein (or a variation thereof) are considered within the scope of theinvention.

In this assay, cells that express the long form of the human or murineOb receptor (or any other long form, i.e., signalling competent,mammalian Ob receptor and harbor a reporter construct that is responsiveto activation of the Ob receptor by leptin are exposed to one or moretest compounds in the presence of leptin. Compounds that act asantagonists of the Ob receptor activity will decrease expression of thereporter construct while compounds that act as agonists of the Obreceptor will increase expression of the reporter gene. In manyinstances, it is desirable to pool test compounds. Thus, 1,000 testcompounds can be divided into 100 pools of 10 compounds. Each poolapplied to cells which expressed Ob receptor and harbor an Ob receptorresponsive reporter gene. The level of reporter gene expression ismeasured and compared to the expression level of otherwise identicalcells which are not exposed to the pool of test compounds. The compoundsin pools that alter reporter expression can then be tested individually.

Cell Culture and Transfection

Immortalized hypothalamic GnRH neurons (for a description of theproduction of the cell line, see Mellon et al., 1990, Neuron 5:1-10) areone type of cell which can be used in this assay. In one example of thescreening assay, these cells are plated on 96-well plaLes, cultureduntil they were approximately 70% confluent, and transfected with a cDNAencoding the long form of the murine or the human Ob receptor(Tartaglia, J. Biol Chem. 272:6093-6096, 1997; the long form of themurine Ob receptor is shown in FIGS. 7A-7B (SEQ ID NO:43 and the longform of the human Ob receptor, including the signal sequence, is shownin FIGS. 3A-3F (SEQ ID NO:4)) and a cDNA reporter construct. Thereporter construct contains a sequence encoding a secreted form ofalkaline phosphatase (SEAP; Clontech), which is placed under the controlof the Ob Receptor responsive promoter element, IL6 RE (describedabove). The constructs are transfected into the cells usingLipofectamine™ (Life Technologies, Gaithersburg, Md.), according to themanufacturer's instructions.

Contacting Cells with Leptin or Potential Agonists or PotentialAntagonists of Ob Receptor Activity

Forty-eight hours after transfection, the cultured cells are washedtwice with serum-free medium, and contacted with either: (1) leptinalone, at a concentration that does not produce maximal stimulation ofthe receptor (as a control), or (2) leptin, at the same concentration asapplied to the control cells, and one or more test compounds, e.g., asmall molecule library (as the experimental group). In both instances,leptin and the compounds are applied to the cells in non-supplementedculture medium. Twenty-four hours later, 100 μl of culture medium areremoved, and SEAP activity is assessed by measuring chemiluminescencewith the Great Escape™ alkaline phosphatase detection kit. The kit ismanufactured by Clontech (Palo Alto, Calif.), and is used according totheir directions. Luminescence values are measured using a Microbetaplus™ liquid scintillation counter (Wallac), and can be expressed asarbitrary units of luminescence activity.

An increase in the activity of the reporter in the presence of a testcompound, compared with activity in the absence of a test compound,indicates the presence of an agonist of Ob receptor activity.Conversely, a decrease in the activity of the reporter in the presenceof a test compound (compared with activity in the absence of a testcompound), indicates the presence of an antagonist of Ob receptoractivity.

The agonist or antagonist may act at any point in the signalling pathwaybetween binding of leptin to Ob receptor and expression of the reportergene. Thus, the screening method is broadly useful for indentifyingcompounds that alter signalling components of the Ob receptor pathway.

Modifications of the assay described above will be apparent to those ofskill in the art. For example, it will be apparent that any moleculethat activates the Ob receptor or the Ob receptor pathway can be used inthe assay described above in the place of leptin; for example, one coulduse a fragment of the leptin molecule that retains the ability to bindto and activate the Ob receptor. Alternatively, antibodies may be usedto activate the receptor. Similarly, when screening compounds for theirability to activate the Ob receptor (i.e., when searching for receptoragonists), the assay can be carried out in the absence of leptin. Inthis case, the expression of the reporter construct can simply becompared in cells that are exposed to a putative agonist (i.e., cellsthat are “treated”) and cells that are not (i.e., cells that are“untreated”). In addition, the cell can be a cell that naturallyexpresses an Ob receptor or that is stably transfected with an Obreceptor-encoding sequence. In events such as these, the assay can beperformed by introducing only a reporter construct into the cell.

In addition to the IL-6 RE, the assay can be conducted using a reportergene that is driven by HRRE (an HRRE-CAT reporter construct is describedabove). Furthermore, the reporter gene itself can vary; reporter genescommonly used by those of skill in the art include β-lactamase,chloramphenicol acetyltransferase (CAT), adenosine deaminase (ADA),aminoglycoside phosphotransferase (neo^(r), G418^(r)), dihydrofolatereductase (DHFR), hygromycin-B-phosphotransferase (HPH), thymidinekinase (TK), lacZ (encoding β-galactosidase), and xanthineguaninephosphoribosyltransferase (XGPRT).

To determine whether the agonist or antagonist is specific for thereceptor itself, a population of cells can be co-transfected asdescribed above, except with a reporter construct and a construct thatencodes a receptor that is related to the Ob receptor by sequencehomology, such as a human G-CSF receptor. As discussed above, the Obreceptor of the invention has amino acid sequence motifs found in theClass I cytokine receptor family, and is most related to the gp130signal transducing component of the IL-6 receptor, the G-CSF receptor,and the LIF receptor. If the activity remains essentially the same asthat observed following co-transfection with the reporter construct anda construct encoding an Ob receptor, the agonist or antagonist isexerting a non-specific effect (i.e., the compound is not specific toonly the Ob receptor).

Similarly, one can determine whether the agonist or antagonist isinteracting with a component of the signalling pathway downstream fromthe Ob receptor (rather than interacting with the receptor itself) byco-transfecting cells with the reporter construct and a construct thatencodes a chimeric polypeptide consisting of the extracellular domain ofthe Ob receptor and an intracellular domain of G-CSF. If the activity ofthe reporter is decreased or increased in cells transfected with thechimera, the respective agonist or antagonist is not specific for the Obreceptor pathway.

Accordingly, the invention features methods for identifying a compounduseful for the treatment of a body weight disorder. One method isperformed by contacting a compound with a cell that contains a reportergene whose expressing is altered as a consequence of activation of theOb receptor. An increase or decrease in expression of the reporter geneindicates the presence of a compound that alters activity of the Obreceptor signallilng pathway, and which is therefore a candidatetherapeutic agent for treatment of a body weight disorder. A secondmethod can be performed by contacting a cell comprising a reporter genethat is expressed following activation of the Ob receptor with (a) thecompound, and (b) an agonist of the Ob receptor. In this case, adecrease in expression of the reporter gene, relative to the level ofexpression when only the agonist is applied, indicates the presence of acompound that antagonizes the Ob receptor, and which is therefore acandidate therapeutic agent for treatment of a body weight disorder.Alternatively, the effect of the compound on reporter gene expression inthe presence of an agnoist of the Ob receptor (e.g., leptin) can bemeasured).

15. Administration of an OBR-Ig Fusion Protein Increases Food Intake

To determine whether an ObR-Ig fusion protein is active whenadministered to animals (i.e., whether it can alter food intake), achimeric protein containing the murine Ob receptor and an immunoglobulinprotein was injected into normal mice and mice that developed anorexiafollowing treatment with endotoxin. This model is described in Grunfeldet al., 1997, J. Clin Invest. 97:2152-2157. For guidance in theconstruction of ObR-immunoglobulin fusion proteins, see Example 10,supra.

Male C57BL/6J mice (14-16 weeks of age) were housed in metabolic cages(2 mice/cage) and provided with standard laboratory rodent chow. Theanimals were allowed to adapt to their environment for several daysbefore beginning the experiment. Lipopolysaccharide (LPS) (E. coli055:B5) was freshly prepared in phosphate buffered saline (PBS) andinjected intraperitoneally (i.p.) at a dose of 10 μg/mouse. The Obreceptor-IgG fusion protein was also prepared in PBS and injectedintravenously at a dose of 150 μg/mouse. In one group of animals, bothLPS and the Ob receptor-IgG fusion protein were administered. In thisgroup, the fusion protein was administered twice; at the same time theLPS was administered, and 24 hours later. Food intake was monitoredevery 24 hours for 4 days by weighing the amount of food remaining inthe cage. The results are shown in FIG. 16, where each data pointrepresents the average food intake of animals in each group. Food intakeby mice that received the Ob receptor-Ig fusion protein was greater thanthe food intake by mice that received PBS. In addition, LPS treated micethat also received the Ob receptor-Ig fusion protein had a greater foodintake than mice that received LPS alone: 72 hours after LPSadministration, the mice that also received the Ob receptor-Ig fusionprotein were eating nearly as well as the control (PBS treated) mice,whereas animals that received LPS without the Ob receptor-Ig fusionprotein still exhibited a 50% reduction in food intake. Therefore,administration of an ObR-Ig fusion protein has been demonstrated tomodulate food intake in vivo in both normal and anorexic mammals.

16. SOCS-1 Interacts with JAK-2 and Inhibits ObR Signaling

A yeast two hybrid screen was used to identify proteins that interactwith the JH1 domain of JAK2, a protein which interacts with ObR. Theidentification of proteins which interact with JAK2 is important becausesuch proteins are likely to play a role in signal transduction initiatedby the binding of leptin to ObR. The JH1 domain of JAK2 is of particularinterest because it is the most C-terminal domain of JAK2 that possesseskinase activity. The results presented in these examples demonstratethat: 1) that two forms of SOCS-1 interact with JAK2; and 2) theexpression of SOCS-1 in cells expressing ObR can inhibit ObR mediatedsignaling.

16.1 Materials and Methods 16.1.1 Cells

Yeast strain HF7c (MATa, ura3-52, his3-200, lys2-801, ade2-101,trp1-901, leu2-3, 112, gal4-542, gal80-538,LYS2::GAL1_(UAS)-GAL1_(TATA)-HIS3,URA3::GAL4_(17mers(x3))-CYC1_(TATA)-lacZ; Feilotter et al., 1994, Nucl.Acids Res. 22:1502-1503) was used in the yeast two hybrid screen.Standard yeast media including synthetic complete medium lackingL-leucine, L-tryptophan, and L-histidine were prepared and yeast geneticmanipulations were performed as described by Sherman (1991, Meth.Enzymol. 194:3-21). Yeast transformations were performed using standardprotocols (Gietz et al., 1992, Nucleic Acids Res. 20:1425; Ito et al.,1983, J. Bacteriol. 153:163-168). Plasmid DNAs were isolated from yeaststrains using standard techniques (Hoffman and Winston, 1987, Gene,57:267-272).

16.1.2 Plasmids

Plasmid pGBT9, a TRP1 amp^(r) vector encoding the DNA binding domain ofGAL4 (amino acids 1-147; Bartel et al., 1993, Cellular Interactions inDevelopment _(—):153-159.), was used to create a plasmid, pRG54, whichencodes a GAL4 DNA binding domain-JAK2 JH1 domain hybrid protein. Tocreate pRG54, DNA encoding amino acids 839-1129 of murine JAK2(Silvennoinen et al., 1993, Proc. Nat. Acad. Sci. USA, 90:8429-8433) wasamplified by PCR and cloned in frame to the portion of pGBT9 encodingthe DNA binding domain of GAL4. Plasmid pRG54 was transformed intotwo-hybrid screening strain HF7c for screening. Plasmid pACTII, whichencodes the activation domain of GAL4 (amino acids 768-881), was used togenerate the library used in the two hybrid screen.

Plasmid pMET7, a mammalian expression vector which utilizes the SRαpromoter (Tartaglia et al., 1995, Cell 83:1263-1271), was used to createplasmid pMET7-SOCS1, a plasmid used to express a portion of murineSOCS-1b, described in greater detail below.

Plasmid pMET7-H60, which encodes the long form of human ObR (Tartagliaet al. (1995) Cell 83:1263-1271), and the STAT-responsive reporter geneconstruct pIL6RE-SEAP (White et al., 1997, J. Biol. Chem. 272:4065-4071)have been described previously.

16.1.3 Two-Hybrid Screening

Two-hybrid screening was carried out essentially as described by Bartelet al. (1993, Cellular Interactions in Development. _(—):153-159.).Briefly, by inserting murine hypothalmic cDNA adjacent to the DNAsequence within pACTII that encodes the GAL4 activation domain, libraryof hybrid proteins was created. Approximately 10⁷ clones from thislibrary were screened by first selecting for clones which are able togrow on synthetic complete medium lacking L-leucine, L-tryptophan, andL-histidine and then using a filter disk beta-galactosidase (beta-gal)assay (Brill et al., 1994, Mol. Biol. Cell, 5:297-312) to identifyclones which encoded potential interacting proteins. Colonies to betested were grown as patches of cells on appropriate medium at 30° C.overnight and then replica plated onto Whatman #50 paper (Schleicher &Schuell, #576) that had been placed on the test medium in petri dishes.After growth overnight at 30° C., the paper disks were removed from theplates and the cells on them were permeabilized by immediately immersingthe paper disks in liquid nitrogen for 30 seconds. The disks were thenthawed at room temperature for 20 seconds and placed in petri dishesthat contained a disk of Whatman #3 paper (Schleicher & Schuell, #593)saturated with 2.5 ml of Z buffer containing 37 μl of 2% weight pervolume of the chromogenic beta-gal substrate X-gal. The disks wereincubated at 30° C. and inspected periodically for the development ofthe blue color diagnostic of beta-gal activity in this assay. The assaywas stopped by removing the paper disk containing the patches of cellsand air drying it.

16.1.4 ObR Signaling Assay

The 293 cells were used in the ObR signaling assay. These cells weregrown in DMEM supplemented with 10% fetal bovine serum. Transfectionswere carried out using lipofectamine (Gibco/BRL; Gaithersburg, Md.). Forthe activity assays, cells were transfected with 1 μg of the pIL6RE-SEAPreporter plasmid, 3 μg of pMET7-H60 encoding ObR receptor, and 3 μg ofpMET7-SOCS1 encoding SOCS-1, as indicated. Forty eight hours aftertransfection the cells were washed twice with serum-free medium and thentreated with leptin for 24 h in non-supplemented cell culture medium.SEAP reporter activity was measured as previously described (White etal., 1997, Proc. Nat. Acad. Sci. USA, 94:10657-10662).

16.2 Results and Discussion 16.2.1 Two Forms of SOCS-1 Interacts withthe JH1 Domain of JAK2

To identify proteins which are part of the ObR signaling pathway, ayeast two hybrid system was used to identify proteins that interact withthe JH1 domain of JAK2.

DNA encoding the JH1 domain of murine JAK2 (amino acids 839-1129) wascloned into pGBT9 to create a plasmid, pRG54, encoding a GAL4DNA-binding domain-JAK2 fusion gene. Yeast HF7c cells transformed withthis construct grew on synthetic complete medium lacking L-tryptophan,but did not grow on synthetic complete medium lacking L-tryptophan andL-histidine. This demonstrates that the GAL4 DNA-binding domain-JAK2fusion does not have intrinsic transcriptional activation activity.

HF7c cells harboring pRG54 and a beta-galactosidase reporter under thecontrol of GAL4 DNA-binding recognition elements were transformed with alibrary of clones composed of mouse hypothalamic cDNA fused to DNAencoding the activation domain of GAL4. Ten million transformants wereobtained. In this screen, clones which contain a mouse cDNA encoding apolypeptide that interacts with the JH1 domain of JAK2 will both grow onsynthetic complete medium lacking L-leucine, L-tryptophan, andL-histidine and express the beta-galactosidase reporter gene. Seventynine such clones were identified, and 39 of these colones werecharacterized further.

The 5′ end of the cDNA insert of each of the 39 clones encodingpotential JAK2 interacting proteins was sequenced. This sequencingrevealed that the cDNA inserts of eight of the 39 clones (clones 11, 12,18, 32, 50, 77, 102, and 115) encoded a portion of murine SOCS-1. Thegenomic sequence of murine SOCS-1 has been published (Schluter et al.,1996, Mol. Reprod. Dev. 43:1-6; Genbank Accession No. Z47352). Thesequence of the 5′ end of the inserts of clones 11, 12, 18, 32, 50, 77,102, and 115 are included in Table 1. The nucleotide numbers in Table 1refer to the genomic SOCS-1 sequence (Genbank Accession No. Z47352).

TABLE 1 SOCS-1 clones Clone 5′ Fusion junction SOCS-1 Splice Form 1112319-12679 SOCS-1b 12 12319-12739 SOCS-1b 18 11873-11957/12467-12827SOCS-1a 32 11873-11957/12467-12857 SOCS-1a 50 11873-11957/12467-12847SOCS-1a 77 11873-11957/12467-12827 SOCS-1a 102 12478-12838 — 11511877-11957/12467/12847 SOCS-1aAnalysis of the fusion junctions and the clones revealed that thevarious clones represented two apparent splice variants of murine SOCS-1(SOCS-1a and SOCS-1b).

SOCS-1a clones contain nucleotides 11873-11957 of the previouslyidentified SOCS-1 genomic clone followed by sequences starting atnucleotide 12467 of the genomic SOCS-1 clone. SOCS-1a is most likely aproduct of splicing an intron between nucleotide 11957 and 12467 of theSOCS-1 genomic clone. Consensus splice donor and acceptor sites arepresent at nucleotides 1158/9 and 12465-7. The SOCS-1b clones begin atnucleotide 12419 of the published genomic sequence, a region that fallswithin the SOCS-1a intron. Neither the SOCS-1a nor the SOCS-1b clonescontain an stop codon upstream of the published SOCS-1 open readingframe, allowing a read-through from the GAL4 activation domain encodingsequences into the SOCS-1 open reading frame. Various SOCS-1 clones havebeen previously identified (Naka et al. (1997) Nature 387:924-929; Starret al. (1997) Nature 387:917-921; and Endo et al. (1997) Nature387:921-924) but the existence of alternate splice forms does not appearto have been recognized.

Co-transformation of clone 11 (which includes has sequences present inSOCS-1b) or clone 102 (which includes sequences present in both SOCS-1aand SOCS-1b) both of which encode a fusion protein comprising a portionof SOCS-1a into yeast HF7c cells along with pGBT9, which encodes theGAL4 DNA-binding domain alone, or pRG54, which encodes the GAL4 DNAbinding domain fused to the JAK2 JH1 domain, revealed that both clone 11and clone 102 encode polypeptides which interact with the JH1 domain ofJAK2, but not the unrelated GAL4 DNA binding domain.

16.2.4 Tissue Distribution of SOCS-1

To determine the relative abundance and tissue distribution of SOCS-1aand SOCS-1b, a poly A⁺ mRNA Northern blot was analyzed with twodifferent probes. The first probe, which corresponds to nucleotides12146-12464 of murine genomic SOCS-1, was designed to recognize SOCS-1b,but not SOCS-1a (the “SOCS-1b probe”). The second probe, whichcorresponds to nucleotides 13228-13504 of murine genomic SOCS-1,recognizes a 3′ untranslated region common to both SOCS-1a and SOCS-1b(the “SOCS-1a/SOCS-1b probe”).

Both the SOCS-1b probe and the SOCS-1a/SOCS-1b probe hybridized to a 1.8kb transcript that was most abundant in spleen and lung. The 1.8 kbtranscript was also observed in all other tissues examined (heart,brain, liver, muscle, kidney and testis). The SOCS-1a/SOCS-1b probe, butnot the SOCS-1b probe, hybridized to a 1.4 kb transcript that was mostabundant in spleen and lung. The 1.4 kb transcript was present at lowerlevels in all other tissues examined (heart, brain, liver, muscle,kidney and testis). In blots probed with the SOCS-1a/SOCS-1b probe, the1.4 kb transcript was much more abundant than the 1.8 kb transcript inall tissues examined. These results suggest that the sizes of theSOCS-1a and SOCS-1b transcripts are 1.4 kb and 1.8 kb respectively, andthat there are no apparent differences between tissues with respect tothe relative amounts of the two transcripts.

16.2.5 SOCS-1 Inhibits ObR-Mediated Signaling

To determine whether SOCS-1 can inhibit ObR-mediated signaling byleptin, 293 cells were cotransfected with a reporter plasmid that isresponsive to STAT activation (pIL6-RE-SEAP) and either: 1) a plasmidwhich expresses the long form of human ObR; or 2) a plasmid whichexpresses the long form of human ObR and a plasmid which expresses aportion of SOCS-1b (nucleotides 12506-13411 of the murine SOCS-1 genomicclone; corresponds to the SOCS-1b portion of clone 11 in Table 1) Thetransfected cells were stimulated with 100 ng/ml leptin for 24 hours.After leptin stimulation, the activity of the pIL-6RE-SEAP reporter wasdetermined by measuring the amount of alkaline phosphatase secreted intothe medium.

Measurement of reporter activity demonstrated that, in the presence ofleptin, expression of the STAT responsive reporter was increasedeight-fold when ObR was also expressed by the cells. However, noactivation of the STAT responsive reporter was observed occurred whenthe cells expressed both ObR and SOCS-1.

Because SOCS-1 inhibits ObR signalling, it may be possible to modulateObR activity by modulating the expression or activity of SOCS-1 or bymodulating the binding of SOCS-1 to JAK2. In addition, one can useSOCS-1 to identify candidate therapeutic compounds which alter theexpression or activity of SOCS-1.

One can identify candidate therapeutic compounds for the treatment of abody weight disorder by providing a cell which expresses a mammalian Obreceptor protein, a mammalian JAK2 protein, and a mammalian SOCS-1protein, and which contains a reporter construct which includes asequence encoding a detectable protein operably linked to an Ob receptorresponsive regulatory element; contacting the cell with a test compound;measuring the expression of the detectable protein in the presence ofthe test compound; and identifying those agents which cause an increaseor a decrease in the expression of the detectable protein. One can alsoidentify candidate therapeutic compounds for the treatment of a bodyweight disorder by: contacting a protein comprising an Ob receptorprotein cytoplasmic domain with a polypeptide which includes the JH1domain of a mammalian JAK2 protein, a mammalian SOCS-1 protein, and atest compound; measuring the binding of the JH1 domain polypeptide tothe SOCS-1 protein in the presence of the test compound; and identifyingagents which increase or a decrease in the binding of the JH1 domainpolypeptide to SOCS-1.

17. Deposit of Microorganisms

The following microorganisms were deposited with the American TypeCulture Collection (ATCC), Rockville, Md., on the dates indicated andwere assigned the indicated accession number:

ATCC Date Microorganism Clone Access. No. of Deposit E. coli strain5312B4F3 famj5312 69952 Nov. 22, 1995 E. coli h-ObRD fahj5312d 69963Dec. 5, 1995 E. Coli h-ObR-p87 h-ObR-p87 69972 Dec. 28, 1995

The present invention is not to be limited in scope by the specificembodiments described herein, which are intended as single illustrationsof individual aspects of the invention, and functionally equivalentmethods and components are within the scope of the invention. Indeed,various modifications of the invention, in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

1. A method for detecting the expression of obese receptor (ObR) in asample comprising: a) contacting the sample with a reagent whichspecifically detects ObR expression; and b) detecting the reagent in thesample, wherein detection of the reagent in the sample indicates thatObR is expressed in the sample.
 2. The method of claim 1, wherein thesample is immobilized.
 3. The method of claim 1, wherein the samplecomprises tissue selected from the group consisting of brain, choroidplexus, hypothalamus, lung and liver.
 4. The method of claim 1, whereinthe reagent specifically detects ObR nucleic acid.
 5. The method ofclaim 4, wherein the sample comprises mRNA molecules and is contactedwith a nucleic acid reagent.
 6. The method of claim 5, wherein thenucleic acid reagent comprises a sequence of 15 to 30 nucleotides of SEQID NO:3 or its complement.
 7. The method of claim 5, wherein the nucleicacid reagent is labeled.
 8. The method of claim 5, wherein the nucleicacid reagent is immobilized.
 9. The method of claim 5, wherein thenucleic acid reagent consists of nucleotides 194-3688 of SEQ ID NO:3 orits complement.
 10. The method of claim 1, wherein the reagentspecifically detects an ObR gene product.
 11. The method of claim 10,wherein the reagent which detects the obR gene product is an obese (Ob)fusion protein.
 12. The method of claim 11, wherein the Ob fusionprotein is labeled.
 13. The method of claim 10, wherein the obR geneproduct comprises amino acid residues 21 to 839 of SEQ ID NO:4.
 14. Themethod of claim 10, wherein the obR gene product comprises amino acidresidues 863 to 1165 of SEQ ID NO:4.
 15. The method of claim 10, whereinthe obR gene product comprises amino acid residues 21 to 1165 of SEQ IDNO:4.
 16. The method of claim 10, wherein the reagent which detects theobR gene product is an antibody directed against an epitope on the obRgene product or an epitope-binding fragment of the antibody.
 17. Themethod of claim 16, wherein the antibody or epitope-binding fragmentthereof is labeled.